Modified pluripotent cells

ABSTRACT

The invention discloses for the first time pluripotent cells, including induced pluripotent stem cells, embryonic stem cells, and hypo-immune pluripotent cells that are ABO blood type O Rhesus Factor negative and evade rejection resulting from blood type antigen mismatch. The invention further provides universally acceptable “off-the-shelf” pluripotent cells and derivatives thereof for generating or regenerating specific tissues and organs.

I. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/846,399, filed May 10, 2019 and U.S.Provisional Application No. 62/855,499, filed May 31, 2019, each ofwhich are incorporated herein by reference in their entirety.

II. FIELD OF THE INVENTION

The invention relates to regenerative cell therapy. In some embodiments,the regenerative cell therapy comprises transplanting cell lines intopatients in need thereof. In some embodiments, the cell lines comprise ORh− hypoimmunogenic pluripotent cells. In some embodiments, theregenerative cell therapy reduces the propensity for the cell transplantrecipient's immune system to reject allogeneic material. In someembodiments, the regenerative cell therapy is used in the treatment ofinjured organs and tissue. In some embodiments, the regenerative celltherapy is used for rehabilitating damaged tissues after myocardialinfarction.

III. BACKGROUND OF THE INVENTION

Regenerative cell therapy is an important potential treatment forregenerating injured organs and tissue. With the low availability oforgans for transplantation and the accompanying lengthy wait, thepossibility of regenerating tissue by transplanting readily availablecell lines into patients is understandably appealing. Regenerative celltherapy has shown promising initial results for rehabilitating damagedtissues after transplantation in animal models (e.g. after myocardialinfarction). The propensity for the transplant recipient's immune systemto reject allogeneic material, however, greatly reduces the potentialefficacy of therapeutics and diminishes the possible positive effectssurrounding such treatments.

Autologous induced pluripotent stem cells (iPSCs) theoreticallyconstitute an unlimited cell source for patient-specific cell-basedorgan repair strategies. Their generation, however, poses technical andmanufacturing challenges and is a lengthy process that conceptuallyprevents any acute treatment modalities. Allogeneic iPSC-based therapiesor embryonic stem cell-based therapies are easier from a manufacturingstandpoint and allow the generation of well-screened, standardized,high-quality cell products. Because pluripotent stem cells can bedifferentiated into any cell type of the three germ layers, thepotential application of stem cell therapy is wide-ranging.Differentiation can be performed ex vivo or in vivo by transplantingprogenitor cells that continue to differentiate and mature in the organenvironment of the implantation site. Ex vivo differentiation allowsresearchers or clinicians to closely monitor the procedure and ensuresthat the proper population of cells is generated prior totransplantation. Because of their allogeneic origin, however, such cellproducts could undergo rejection.

The art seeks stem cells capable of producing cells that are used toregenerate or replace diseased or deficient cells. Pluripotent stemcells (PSCs) may be used because they rapidly propagate anddifferentiate into many possible cell types. The family of PSCs includesseveral members generated via different techniques and possessingdistinct immunogenic features. Patient compatibility with engineeredcells or tissues derived from PSCs determines the risk of immunerejection and the requirement for immunosuppression.

Embryonic stem cells (ESCs) isolated from the inner cell mass ofblastocysts can exhibit the histocompatibility antigens that aremismatches with recipients. This immunological barrier cannot be solvedby human leukocyte antigen (HLA)-typed banks of ESCs because evenHLA-matched PSC grafts undergo rejection because of mismatches innon-HLA molecules that function as minor antigens. This is also true forallogeneic induced pluripotent stem cells (iPSCs).

To circumvent the problem of rejection, different techniques for thegeneration of patient-specific pluripotent stem cells have beendeveloped. These include the transfer of a somatic cell nucleus into anenucleated oocyte (somatic cell nucleus transfer (SCNT) stem cells), thefusion of a somatic cell with an ESC (hybrid cell), and the reprogramingof somatic cells using certain transcription factors (induced PSCs oriPSCs). SCNT stem cells and iPSCs, however, may have immuneincompatibilities with the nucleus or cell donor, respectively, despitechromosomal identity. SCNT stem cells carry mitochondrial DNA (mtDNA)passed along from the oocyte. mtDNA-coded proteins can act as relevantminor antigens and trigger rejection. DNA and mtDNA mutations andgenetic instability associated with reprograming and culture-expansionof iPSCs can also create minor antigens relevant for immune rejection.This hurdle decreases the likelihood of successful, large-scaleengineering of compatible patient-specific tissues using SCNT stem cellsor iPSCs.

More recently, the advent of hypoimmune pluripotent (HIP) cells thatevade rejection by the host allogeneic immune system has resulted in amajor breakthrough for allogeneic transplantations. See WO 2018/132783.These cells are engineered to reduce HLA-I and HLA-II expression toavoid initiation of an immune response and to increase production ofCD47 to suppress phagocytic innate immune surveillance. The inventionsdescribed herein build on this technology and further reduce HIP cellrejection.

IV. SUMMARY OF THE INVENTION

A previously unrecognized cause of rejection of transplanted cells hasnow been linked to blood group antigens. Blood products can beclassified into different groups according to the presence or absence ofantigens on the surface of every red blood cell in a person's body (ABOBlood Type). The A, B, AB, and A1 antigens are determined by thesequence of oligosaccharides on the glycoproteins of erythrocytes. Thegenes in the blood group antigen group provide instructions for makingantigen proteins. Blood group antigen proteins serve a variety offunctions within the cell membrane of red blood cells. These proteinfunctions include transporting other proteins and molecules into and outof the cell, maintaining cell structure, attaching to other cells andmolecules, and participating in chemical reactions.

The Rhesus Factor (Rh) blood group is the second most important bloodgroup system, after the ABO blood group system. The Rh blood groupsystem consists of 49 defined blood group antigens, among which fiveantigens, D, C, c, E, and e, are the most important. Rh(D) status of anindividual is normally described with a positive or negative suffixafter the ABO type. The terms “Rh factor,” “Rh positive,” and “Rhnegative” refer to the Rh(D) antigen only. Antibodies to Rh antigens canbe involved in hemolytic transfusion reactions and antibodies to theRh(D) and Rh(c) antigens confer significant risk of hemolytic disease ofthe fetus and newborn. ABO antibodies develop in early life in everyhuman. However, rhesus antibodies in Rh− humans develop only when theperson is sensitized. This occurs by giving birth to a rh+ baby or byreceiving an Rh+ blood transfusion.

This invention provides ABO blood type O and/or Rhesus Factor negative(O−) populations of pluripotent (PSCO−) cells suitable for transplantionand/or differentiation. The PSCO− cells include induced iPSCs (iPSCO−),embryonic ESCs (ESCO−), and cells differentiated from those cells,including O− endothelial cells, O− cardiomyocytes, O− hepatocytes, O−dopaminergic neurons, O− pancreatic islet cells, O− retinal pigmentendothelium cells, and other O− cell types used for transplantation andmedical therapies. These would include O− chimeric antigen receptor(CAR) cells, such as CAR-T cells, CAR-NK cells, and other engineeredcell populations. In some embodiments, the cells are not hematopoieticsstem cells. The invention further provides universally acceptable“off-the-shelf” ESCO-s and PSCO-s and derivatives thereof for generatingor regenerating specific tissues and organs.

Another aspect of the invention provides methods of generatingpopulations of PSCO−, iPSCO−, ESCO− and other O− cells fortransplantation. The invention also provides methods of treatingdiseases, disorders, and conditions that benefit from thetransplantation of pluripotent or differentiated cells.

In some embodiments of the invention, the ABO blood group type O resultsfrom a reduced ABO blood group protein expression. In other aspects, theABO blood group is endogenously type O. In some aspects of theinvention, the HIPO− cell has an ABO blood group type O that resultsfrom a disruption in human Exon 7 of the ABO gene. In some embodiments,both alleles of Exon 7 of the ABO gene are disrupted. In someembodiments, the disruption in both alleles of Exon 7 of the ABO generesults from a Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/Cas9 reaction that disrupts both of the alleles.

In other aspects, the ABO blood group type O results from an enzymaticmodification of an ABO gene product on a surface of the cell. In apreferred aspect, the enzymatic modification removes a carbohydrate fromthe ABO gene product. In another preferred aspect, the enzymaticmodification removes a carbohydrate from an ABO A1 antigen, A2 antigen,or B antigen.

In some embodiments of the invention, the Rh blood group is endogenouslytype Rh−. In another aspect, the Rh− blood group results from reducingor eliminating Rh protein expression. In another aspect, the type Rh−results from disrupting the gene encoding Rh C antigen, Rh E antigen,Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd(JK) Jkb antigen, or/and or Kidd SLC14A1. In some embodiments thedisruption results from a CRISPR/Cas9 reaction that disrupts bothalleles of the gene encoding Rh C antigen, Rh E antigen, Kell K antigen(KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen,or/and or Kidd SLC14A1.

In some embodiments of the invention, the O− cells (e.g., PSCO−, iPSCO−,ESCO− and cells derived therefrom) of the invention are of mammalianorigin, for example, human, bovine, porcine, chicken, turkey, horse,sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama,alpaca, mouse, rat, dog, cat, hamster, or guinea pig origin.

In a specific embodiment, the invention provides hypoimmune pluripotentABO blood type O Rhesus Factor negative (HIPO−) cells that evaderejection by the host allogeneic immune system and avoid blood antigentype rejection. In some embodiments, the HIPO− cells are engineered toreduce or eliminate HLA-I and HLA-II expression, increase expression ofan endogenous protein that reduces the susceptibility of the pluripotentcell to macrophage phagocytosis, and comprise a universal blood group ORh− (“O−”) blood type. The universal blood type may be achieved byeliminating ABO blood group A and B antigens and Rh factor expression,or by starting with an O− cell line. These novel HIPO− cells evade hostimmune rejection because they have an impaired antigen presentationcapacity, protection from innate immune clearance, and lack blood grouprejection.

In certain embodiments, HIPO− cells of the invention comprise: anendogenous Major Histocompatibility Antigen Class I (HLA-I) functionthat is reduced when compared to an unmodified pluripotent cell; anendogenous Major Histocompatibility Antigen Class II (HLA-II) functionthat is reduced when compared to an unmodified pluripotent cell;increased expression of CD47 as compared to an unmodified pluripotentcell that reduces susceptibility to NK cell killing; an ABO blood grouptype O (O); and a Rhesus Factor (Rh) blood type negative (−); whereinthe human hypoimmunogenic pluripotent O− (HIPO−) cell is lesssusceptible to rejection when transplanted into a subject when comparedwith an otherwise similar hypoimmunogenic pluripotent (HIP) cell that isan ABO blood group or Rh factor mismatch to the subject.

In certain embodiments of the invention, the HIPO− cell has a reducedHLA-I function by virtue of a reduction in β-2 microglobulin proteinexpression. In another aspect, a gene encoding the β-2 microglobulinprotein is knocked out. In some embodiments, both alleles of the MB2gene are disrupted. In some embodiments, that disruption results from aCRISPR/Cas9 reaction. In some embodiments of the invention, the HIPO−cell has a reduced HLA-I function by virtue of a reduction in HLA-Aprotein expression. In another aspect, a gene encoding the HLA-A proteinis knocked out. In another embodiment, the HLA-I function is reduced bya reduction in HLA-B protein expression. In some embodiments, a geneencoding the HLA-B protein is knocked out. In other embodiments of theinvention, the HIPO− cell has a reduced HLA-I function by virtue of areduction in HLA-C protein expression. In some embodiments, a geneencoding the HLA-C protein is knocked out. In some embodiments, theHIPO− cell does not comprise an HLA-I function.

In certain embodiments of the invention, the HIPO− cell has a reducedHLA-II function by virtue of a reduction in CIITA protein expression. Inanother embodiment, a gene encoding the CIITA protein is knocked out. Inan aspect of the invention, the HIPO− cell has a reduced HLA-II functionby virtue of a reduction in HLA-DP protein expression. In anotheraspect, a gene encoding the HLA-DP protein is knocked out. In anotheraspect, the HLA-II function is reduced by a reduction in HLA-DR proteinexpression. In another aspect, a gene encoding the HLA-DR protein isknocked out. In another aspect, the HLA-II function is reduced by areduction in HLA-DQ protein expression. In another aspect, a geneencoding the HLA-DQ protein is knocked out. In another aspect, thehypoimmunogenic pluripotent cell does not comprise an HLA-II function.

In some embodiments, the HIPO− cells of the invention are engineered tohave increased expression of a protein that reduces the susceptibilityof the pluripotent cell to macrophage phagocytosis. In some embodiments,the increased expression results from a modification to an endogenousgene locus. In some embodiments, the HIPO− cells have a reducedsusceptibility to NK cell killing resulting from an increased expressionof a CD47 protein. In another aspect, the increased CD47 proteinexpression results from a modification to an endogenous CD47 gene locus.In another aspect, the increased CD47 protein expression results fromaddition of a CD47 transgene. In some embodiments, the increased CD7protein expression results from introducing at least one copy of a humanCD47 gene under the control of a promoter into the cell. In a preferredaspect, the promoter is a constitutive promoter.

In some embodiments, the HIPO− cells of the invention further comprise asuicide gene that is activated by a trigger that causes thehypoimmunogenic pluripotent cell to die. In another aspect, the suicidegene is a herpes simplex virus thymidine kinase gene (HSV-tk) and thetrigger is ganciclovir. In another aspect, the suicide gene is anEscherichia coli cytosine deaminase gene (EC-CD) and the trigger is5-fluorocytosine (5-FC). In another aspect, the suicide gene encodes aninducible Caspase protein and the trigger is a chemical inducer ofdimerization (CID). In another more preferred aspect, the CID is AP1903.

One aspect of the invention, provides cells derived from the PSCO−,iPSCO−, ESCO−, and or HIPO− cells described herein, wherein the cellsare selected from the group consisting of a chimeric antigen receptor(CAR) cell, an endothelial cell, a dopaminergic neuron, a pancreaticislet cell, and a retinal pigment endothelium cell. In a preferredaspect, the CAR cell is a CAR-T cell.

The invention further provides a method of treating a disease, disorder,or condition that can benefit from transplantation with the cells of theinvention or derivatives thereof. The method comprises administeringPSCO−, iPSCO−, ESCO−, HIPO− and/or other ABO− cells as described herein.In one aspect, the PSCO−, iPSCO−, ESCO−, HIPO− and/or other ABO− cellsare selected from the group consisting of chimeric antigen receptor(CAR) cells, endothelial cells, dopaminergic neurons, pancreatic isletcells, and retinal pigment endothelium cells. In some embodiments, thedisease is selected from the group consisting of Type I Diabetes,cardiac diseases, neurological diseases, cancers, ocular diseases, andvascular diseases. In some embodiments of the invention, the methodcomprises transplanting ABO− cells of the invention, including PSCO−,iPSCO−, ESCO−, HIPO− cells and cells derived from PSCO−, iPSCO−, ESCO−,and HIPO− cells into a mammalian subject. In some embodiments, thesubject is a human, cow, pig, chicken, turkey, horse, sheep, goat,donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse,rat, dog, cat, hamster, guinea pig.

Another aspect of the invention provides a method for generating ahypoimmunogenic pluripotent ABO group O Rh factor negative (HIPO−) cellsfrom an O− iPSCs comprising: (a) eliminating the MajorHistocompatibility Antigen Class I (HLA-I) function or reducing HLA-Ifunction as compared to an unmodified pluripotent cell; (b) eliminatingthe Major Histocompatibility Antigen Class II (HLA-I) function orreducing HLA-I function as compared to an unmodified pluripotent cell;(c) increasing the expression of CD47 as compared to expression of CD47in an unmodified iPSC. In some embodiments, the starting iPSC is not O−and thus the method further comprises: (d) eliminating any ABO bloodgroup antigen to provide ABO blood type O; and (e) eliminating anyRhesus Factor (Rh) blood group antigen to provide Rh type negative (−).

The invention also provides one or more cells that are derived ordifferentiated from the induced ABO blood group O− Rh factor (−)pluripotent (e.g., PSCO−, iPSCO, ESCO−) cells described herein. Thesecells comprise an ABO blood group type O (O) and a Rhesus Factor (Rh)blood type negative (−), rendering them less susceptible to rejectionwhen transplanted into a subject when compared with cells that are ABOblood group or Rh factor mismatched to the subject.

The invention also provides for the screening and/or stratifying ofsubjects for administration of hypoimmunogenic cells, wherein the ABOblood group type of the hypoimmunogenic cells is matched with thesubject's ABO blood group type prior to the administration of the cells.For example, the invention may provide for the administration of ABOblood group type A hypoimmunogenic cells to a subject who is determinedto be ABO blood group type A or AB; the administration of ABO blood typeB hypoimmunogenic cells to a subject who is determined to be ABO bloodgroup type B or AB; the administration of ABO blood group type ABhypoimmunogenic cells to a subject who is determined to be ABO bloodgroup type AB; or the administration of ABO blood group type Ohypoimmunogenic cells to a subject who is determined to be ABO bloodgroup type A, B, AB, or O.

Another aspect of the invention provides for the screening and/orstratifying of subjects for administration of hypoimmunogenic cells,wherein the ABO blood group type and the Rh blood type of thehypoimmunogenic cells are matched with the subject's ABO blood grouptype and Rh blood type prior to the administration of the cells. Forexample, the invention may provide for the administration of ABO bloodgroup type A, Rh positive (+) hypoimmunogenic cells to a subject who isdetermined to be ABO blood group type A or AB and Rh positive (+). Inanother example, the invention may provide for the administration of ABOblood group type B, Rh positive (+) hypoimmunogenic cells to a subjectwho is determined to be ABO blood group type B or AB and Rh positive(+). In another example, the invention may provide for theadministration of ABO blood group type AB, Rh positive (+)hypoimmunogenic cells to a subject who is determined to be ABO bloodgroup type AB and Rh positive (+). In another example, the invention mayprovide for the administration of ABO blood group type O, Rh positive(+) hypoimmunogenic cells to a subject who is determined to be ABO bloodgroup type A, B, AB or O, and Rh positive (+). In another example, theinvention may provide for the administration of ABO blood group type A,Rh negative (−) hypoimmunogenic cells to a subject who is determined tobe ABO blood group type A or AB and Rh negative (−). In another example,the invention may provide for the administration of ABO blood group typeB, Rh negative (−) hypoimmunogenic cells to a subject who is determinedto be ABO blood group type B or AB and Rh negative (−). In anotherexample, the invention may provide for the administration of ABO bloodgroup type AB, Rh negative (−) hypoimmunogenic cells to a subject who isdetermined to be ABO blood group type AB and Rh negative (−). In anotherexample, the invention may provide for the administration of ABO bloodgroup type O, Rh negative (−) hypoimmunogenic cells to a subject who isdetermined to be ABO blood group type A, B, AB or O, and Rh negative(−).

Another aspect of the invention provides for the screening and/orstratifying of subjects for administration of hypoimmunogenic cells,wherein the ABO blood group type of the hypoimmunogenic cells arematched with the subject's ABO blood group type, but the Rh blood typeof the hypoimmunogenic cells and the Rh blood type of the subject arenot matched. In this aspect, the cells are only administered if they areRh blood type negative (−) and they are administered to a subject who isRh blood type positive (+). For example, the invention may provide forthe administration of ABO blood group type A, Rh negative (−)hypoimmunogenic cells to a subject who is determined to be ABO bloodgroup type A or AB and Rh positive (+). In another example, theinvention may provide for the administration of ABO blood group type B,Rh negative (−) hypoimmunogenic cells to a subject who is determined tobe ABO blood group type B or AB and Rh positive (+). In another example,the invention may provide for the administration of ABO blood group typeAB, Rh negative (−) hypoimmunogenic cells to a subject who is determinedto be ABO blood group type AB and Rh positive (+). In another example,the invention may provide for the administration of ABO blood group typeO, Rh negative (−) hypoimmunogenic cells to a subject who is determinedto be ABO blood group type A, B or AB, and Rh positive (+).

V. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that adaptive and innate immune cells were not activatedin immune assays using blood from Macaque Rhesus monkeys in whichhypoimmunogenic B2M−/− CIITA−/− rhesus CD47 tg endothelial cells did notsurvive. Hypoimmunogenic B2M−/− CIITA−/− rhesus CD47 tg endothelialcells were not rejected by T cells, Cytotoxic T cells, or NK cells. FIG.1B shows that hypoimmunogenic B2M−/− CIITA−/− rhesus CD47 tg endothelialcells did not trigger antibody production and were not rejected bymacrophages. Because the cells were cleared from the monkeys, theseresults suggested another mechanism for cell death.

FIG. 2A shows that adaptive immune cells (T cells, Cytotoxic T cells,and B cells) were activated in immune assays using blood from MacaqueRhesus monkeys that rejected unmodified human iPSC-derived endothelialcells. FIG. 2B shows that innate immune cells (NK cells and macrophages)were activated in immune assays using blood from Macaque Rhesus monkeysthat rejected HLA-I deficient/HLA-II deficient human iPSC-derivedendothelial cells. FIG. 2C shows that Rhesus Monkey Blood Type B SerumCauses Complement-Dependent Cytotoxicity (CDC) of wild-type (wt) inducedendothelial cells (iECs) of blood type A. FIG. 2D shows that iECs ofblood type O were unaffected. FIG. 2E shows that embryonic stemcell-derived ECs undergo the same ABO blood type-dependent CDC asiPSC-derived iECs.

FIG. 3: Targeted cell killing by ABO blood type-incompatible serum wasconfirmed by incubating human B2M−/− CIITA−/− rhesus CD47 tghypoimmunogenic endothelial cells with rhesus macaque serum. When humanhypoimmunogenic endothelial cells (blood type A) are incubated withrhesus macaque serum (blood type B), cells are killed immediately.Depletion of either IgM or IgG antibodies demonstrated that theABO-antibodies were from the IgM type.

FIG. 4 shows that the human B2M−/− CIITA−/− rhesus CD47 tg cells werenot rejected by other pre-formed antibodies when transplanted across thexenogeneic barrier. Human B2M−/− CIITA−/− rhesus CD47 tg iPSC-derivedendothelial cells (blood type A) were killed when incubated with ABO−mismatched rhesus macaque serum (blood type B). When serum from rhesusmacaque with blood type AB was used, however, the human cells survived.

FIG. 5A shows that human hypoimmunogenic iPSC-derived cardiomyocytes(blood type A) survive when incubated with allogeneic human serum bloodtype A and AB. Serum containing pre-formed antibodies against A (bloodtype O and B), however, killed the cells immediately. FIG. 5B shows thatmature cardiomyocytes (blood type A) survive when incubated with ABOmatched allogeneic human serum (blood type A and AB), but they arekilled when incubated with serum containing pre-formed antibodiesagainst A (blood type O and B).

FIG. 6 shows that blood type A human hepatocytes survive when incubatedwith ABO matched serum (blood type A and AB) but are killed whenincubated with ABO mismatched serum (blood type B and O). Similarly,blood type AB human hepatocytes survive when incubated with ABO matchedserum (blood type AB) but are killed when incubated with ABO mismatchedserum (blood type A, B, and O).

FIG. 7A shows that endothelial cells (blood type A Rh+ or Rh−) are ableto survive when incubated with an ABO matched serum (blood type A) whichis either Rh+ or Rh− when the serum has not been previously sensitizedto the Rh factor. FIG. 7B shows that endothelial cells (blood type A orB Rh+) are killed when incubated with an ABO-compatible serum (bloodtype AB) that is Rh− when the serum contains anti-Rh antibodies due to aprevious sensitization to the Rh factor. Endothelial cells (blood type ARh−) are not killed when incubated with the same blood type AB serumthat contains anti-Rh antibodies because the EC cells don't express theRh antigen. Hypoimmunogenic endothelial cells (B2M−/− CIITA−/− CD47 tg)(blood type A Rh+) are also killed when incubated with an ABO-compatibleserum (blood type AB) that is Rh− and that has been previouslysensitized to the Rh factor. FIG. 7C shows that endothelial cells thatare blood type O Rh+ are killed when using O Rh− serum that has beenpreviously sensitized to the Rh factor. FIG. 7D shows that whenH9-derived ECs of blood type A Rh+ were incubated with ABO-compatibleserum containing Rh antibodies, they underwent CDC killing. FIG. 7Eshows that HEK293 cells incubated with serum containing Rh antibodiesdid not undergo killing.

VI. DETAILED DESCRIPTION OF THE INVENTION

The invention provides Rhesus Factor negative pluripotent cells of anyABO blood type, such as blood type O, that avoid or minimize host immuneresponses due to one or more genetic or enzymatic manipulations asoutlined herein. The cells lack major blood group and immune antigensthat trigger immune responses and may also be engineered to avoidrejection, phagocytosis, or killing. This allows the derivation of“off-the-shelf” cell products for generating specific tissues andorgans. The use of human allogeneic PSCO−, iPSCO, ESCO−, or HIPO− cellsand their derivatives in human patients provides significant benefits,including the ability to avoid cell transplant rejection withoutrequiring long-term adjunct immunosuppressive therapy and drug usegenerally seen in allogeneic transplantations. It also providessignificant cost savings as cell therapies can be used without requiringindividual treatments for each patient. Recently, it was shown that cellproducts generated from autologous cell sources may become subject toimmune rejection with few or even one single antigeneic mutation. Thus,autologous cell products are not inherently non-immunogenic. Also,individual patient cell engineering and quality control is very laborand cost intensive and thus, autologous cells are rarely available foracute treatment options. Allogeneic cell products will be able to beused for a bigger patient population only if the immune hurdle can beovercome. The PSCO−, iPSCO, ESCO−, and HIPO− cells of the invention andcells derived from those cells will provide a universal cell source forthe generation of universally-acceptable derivatives.

In addition to an O− blood type, the present invention, in part,exploits the fetomaternal tolerance that exists in pregnant women.Although half of a fetus' human leukocyte antigens (HLA) are paternallyinherited and the fetus expresses major HLA mismatched antigens, thematernal immune system does not recognize the fetus as an allogeneicentity and does not initiate an immune response, e.g. as is seen in a“host versus graft” type of immune reaction. Fetomaternal tolerance ismainly mediated by syncytiotrophoblast cells in the fetal-maternalinterface. Syncytiotrophoblast cells show little or no proteins of themajor histocompatibility complexes I and II (MHC-I and MHC-II), as wellas increased expression of CD47, known as the “don't eat me” proteinthat suppresses phagocytic innate immune surveillance and elimination ofHLA-devoid cells. Surprisingly, the same tolerogenic mechanisms thatprevent rejection of the fetus during pregnancy also allow the HIPO−cells of the invention to escape rejection and facilitate long-termsurvival and engraftment of these cells after allogeneictransplantation.

Fetomaternal tolerance can be introduced with as little as three geneticmodifications (as compared to unmodified iPSCs, e.g. hiPSCs): tworeductions in activity (“knock outs” as further described herein) andone increase in activity (a “knock in” as described herein). Generally,others have attempted to suppress immunogenicity of iPSCs but have beenonly partially successful; see Rong et al., Cell Stem Cell 14:121-130(2014) and Gornalusse et al., Nature Biotech doi:10.1038/nbt.3860),WO2018/132783 and U.S. Prov. App. Nos. 62/698,941, 62/698,965,62/698,973, 62/698,978, 62/698,981, and 62/698,984, each of which areincorporated by reference herein in their entirety.

Autologous induced pluripotent stem cells (iPSCs) constitute anunlimited cell source for patient-specific, autologous cell-based organrepair strategies. As noted above, however, generation and subsequentdifferentiation of autologous IPSCs into tissue cells pose technical andmanufacturing challenges and are lengthy processes that preclude theiruse in acute treatment modalities. These shortcomings can only beovercome with availability of prefabricated ready-to-use cell or tissueproducts of allogeneic origin.

The starter cell line for prefabricated ready-to-use cell or tissueproducts of allogeneic origin, must be “universal”. The PSCO−, iPSCO,ESCO−, or HIPO− cells of the invention provide for the first time,readily accessible non-immunogenic pluripotent cells for maintenance,differentiation into desired cell and tissue types, and ultimatelytransplantation of their derivatives into patients in need thereof.

Definitions

The term “pluripotent cells” refers to cells that can self-renew andproliferate while remaining in an undifferentiated state and that can,under the proper conditions, be induced to differentiate intospecialized cell types. The term “pluripotent cells,” as used herein,encompass embryonic stem cells and other types of stem cells, includingfetal, amniotic, or somatic stem cells. Exemplary human stem cell linesinclude the H9 human embryonic stem cell line. Additional exemplary stemcell lines include those made available through the National Institutesof Health Human Embryonic Stem Cell Registry and the Howard HughesMedical Institute HUES collection (as described in Cowan, C. A. et. al,New England J. Med. 350:13. (2004), incorporated by reference herein inits entirety).

“Pluripotent stem cells” as used herein have the potential todifferentiate into any of the three germ layers: endoderm (e.g. thestomach linking, gastrointestinal tract, lungs, etc), mesoderm (e.g.muscle, bone, blood, urogenital tissue, etc) or ectoderm (e.g. epidermaltissues and nervous system tissues). The term “pluripotent stem cells,”as used herein, also encompasses “induced pluripotent stem cells”, or“iPSCs”, a type of pluripotent stem cell derived from a non-pluripotentcell. Examples of parent cells include somatic cells that have beenreprogrammed to induce a pluripotent, undifferentiated phenotype byvarious means. Such “iPS” or “iPSC” cells can be created by inducing theexpression of certain regulatory genes or by the exogenous applicationof certain proteins. Methods for the induction of iPS cells are known inthe art and are further described below. (See, e.g., Zhou et al., StemCells 27 (11): 2667-74 (2009); Huangfu et al., Nature Biotechnol. 26(7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); andZhou et al., Cell Stem Cell 8:381-384 (2009); each of which isincorporated by reference herein in their entirety.) The generation ofinduced pluripotent stem cells (iPSCs) is outlined below. As usedherein, “hiPSCs” are human induced pluripotent stem cells, and “miPSCs”are murine induced pluripotent stem cells.

Several characteristics of pluripotent stem cells distinguish them fromother cells. For example, the ability to give rise to progeny that canundergo differentiation, under the appropriate conditions, into celltypes that collectively demonstrate characteristics associated with celllineages from all of the three germinal layers (endoderm, mesoderm, andectoderm) is a pluripotent stem cell characteristic. Expression ornon-expression of certain combinations of molecular markers are alsopluripotent stem cell characteristics. For example, human pluripotentstem cells express at least several, and in some embodiments, all of themarkers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60,TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, andNanog. Cell morphologies associated with pluripotent stem cells are alsopluripotent stem cell characteristics. Cells do not need to pass throughpluripotency to be reprogrammed into endodermal progenitor cells and/orhepatocytes.

As used herein, “multipotent” or “multipotent cell” refers to a celltype that can give rise to a limited number of other particular celltypes. For example, induced multipotent cells are capable of formingendodermal cells. Additionally, multipotent blood stem cells candifferentiate itself into several types of blood cells, includinglymphocytes, monocytes, neutrophils, etc.

As used herein, the term “oligopotent” refers to the ability of an adultstem cell to differentiate into only a few different cell types. Forexample, lymphoid or myeloid stem cells are capable of forming cells ofeither the lymphoid or myeloid lineages, respectively.

As used herein, the term “unipotent” means the ability of a cell to forma single cell type. For example, spermatogonial stem cells are onlycapable of forming sperm cells.

As used herein, the term “totipotent” means the ability of a cell toform an entire organism. For example, in mammals, only the zygote andthe first cleavage stage blastomeres are totipotent.

As used herein, “non-pluripotent cells” refer to mammalian cells thatare not pluripotent cells. Examples of such cells include differentiatedcells as well as progenitor cells. Examples of differentiated cellsinclude, but are not limited to, cells from a tissue selected from bonemarrow, skin, skeletal muscle, fat tissue and peripheral blood.Exemplary cell types include, but are not limited to, fibroblasts,hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells.The starting cells employed for generating the induced multipotentcells, the endodermal progenitor cells, and the hepatocytes can benon-pluripotent cells.

Differentiated cells include, but are not limited to, multipotent cells,oligopotent cells, unipotent cells, progenitor cells, and terminallydifferentiated cells. In particular embodiments, a less potent cell isconsidered “differentiated” in reference to a more potent cell.

A “somatic cell” is a cell forming the body of an organism. Somaticcells include cells making up organs, skin, blood, bones and connectivetissue in an organism, but not germ cells.

Cells can be from, for example, human or non-human mammals. Exemplarynon-human mammals include, but are not limited to, mice, rats, cats,dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, andnon-human primates. In some embodiments, a cell is from an adult humanor non-human mammal. In some embodiments, a cell is from a neonatalhuman, an adult human, or non-human mammal.

As used herein, the terms “subject” or “patient” refers to any animal,such as a domesticated animal, a zoo animal, or a human. The “subject”or “patient” can be a mammal like a dog, cat, bird, livestock, or ahuman. Specific examples of “subjects” and “patients” include, but arenot limited to, individuals (particularly human) with a disease ordisorder related to the liver, heart, lung, kidney, pancreas, brain,neural tissue, blood, bone, bone marrow, and the like.

Mammalian cells can be from humans or non-human mammals. Exemplarynon-human mammals include, but are not limited to, mice, rats, cats,dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, andnon-human primates (e.g., chimpanzees, macaques, and apes).

By “hypoimmunogenic pluripotent” cell or “HIP” cell herein is meant apluripotent cell that retains its pluripotent characteristics and yetgives rise to a reduced immunological rejection response whentransferred into an allogeneic host. In preferred embodiments, HIP cellsdo not give rise to an immune response. Thus, “hypoimmunogenic” refersto a significantly reduced or eliminated immune response when comparedto the immune response of an unmodified or wild type (i.e. “wt”)pluripotent cell, i.e, a cell prior to immunoengineering as outlinedherein. In many cases, the HIP cells are immunologically silent and yetretain pluripotent capabilities. Assays for HIP characteristics areoutlined below.

By “hypoimmunogenic pluripotent cell O−” “hypoimmunogenic pluripotentORh−” cell or “HIPO−” cell herein is meant a HIP cell that is also ABOblood group O and Rhesus Factor Rh−. HIPO− cells may have been generatedfrom O− cells, enzymatically modified to be O−, or geneticallyengineered to be O−.

By “HLA” or “human leukocyte antigen” complex herein is meant a genecomplex encoding the major histocompatibility complex (MHC) proteins inhumans. These cell-surface proteins that make up the HLA complex areresponsible for the regulation of the immune response to antigens. Inhumans, there are two MHCs, class I and class II, “HLA-I” and “HLA-II”.HLA-I includes three proteins, HLA-A, HLA-B and HLA-C, which presentpeptides from the inside of the cell, and antigens presented by theHLA-I complex attract killer T-cells (also known as CD8+ T-cells orcytotoxic T cells). The HLA-I proteins are associated with 13-2microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM,HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cellto T lymphocytes. This stimulates CD4+ cells (also known as T-helpercells). It should be understood that the use of either “MHC” or “HLA” isnot meant to be limiting. Thus, as it relates to mammalian cells, theseterms may be used interchangeably herein.

By “gene knock out” herein is meant a process that renders a particulargene inactive in the host cell in which it resides, resulting either inno protein of interest being produced or an inactive form. As will beappreciated by those in the art and further described below, this can beaccomplished in a number of different ways, including removing nucleicacid sequences from a gene, or interrupting the sequence with othersequences, altering the reading frame, or altering the regulatorycomponents of the nucleic acid. For example, all or part of a codingregion of the gene of interest can be removed or replaced with“nonsense” sequences, all or part of a regulatory sequence such as apromoter can be removed or replaced, translation initiation sequencescan be removed or replaced, etc.

By “gene knock in” herein is meant a process that adds a geneticfunction to a host cell. This causes increased levels of the encodedprotein. As will be appreciated by those in the art, this can beaccomplished in several ways, including adding one or more additionalcopies of the gene to the host cell or altering a regulatory componentof the endogenous gene increasing expression of the protein is made.This may be accomplished by modifying the promoter, adding a differentpromoter, adding an enhancer, or modifying other gene expressionsequences.

“β-2 microglobulin” or “β2M” or “B2M” protein refers to the human β2Mprotein that has the amino acid and nucleic acid sequences shown below;the human gene has accession number NC 000015.10:44711487-44718159.

“CD47 protein” protein refers to the human CD47 protein that has theamino acid and nucleic acid sequences shown below; the human gene hasaccession number NC 000016.10:10866208-10941562.

“CIITA protein” protein refers to the human CIITA protein that has theamino acid and nucleic acid sequences shown below; the human gene hasaccession number NC_000003.12:108043094-108094200.

By “wild type” in the context of a cell herein is meant a cell found innature. However, in the context of a pluripotent stem cell, as usedherein, it also means an iPSC that may contain nucleic acid changesresulting in pluripotency but did not undergo the gene editingprocedures of the invention to achieve hypoimmunogenicity.

By “syngeneic” herein is meant the genetic similarity or identity of ahost organism and a cellular transplant where there is immunologicalcompatibility; e.g. no immune response is generated.

By “allogeneic” herein is meant the genetic dissimilarity of a hostorganism and a cellular transplant where an immune response isgenerated.

By “B2M−/−” herein is meant a diploid cell that has had the B2M geneinactivated in both chromosomes. As described herein, this can be donein a variety of ways.

By “CIITA−/−” herein is meant a diploid cell that has had the CIITA geneinactivated in both chromosomes. As described herein, this can be donein a variety of ways.

By “CD47 tg” (standing for “transgene”) or “CD47+”) herein is meant thatthe host cell expresses CD47 in some cases by having at least oneadditional copy of the CD47 gene.

An “Oct polypeptide” refers to any of the naturally-occurring members ofthe Octamer family of transcription factors, or variants thereof. Theymaintain a similar transcription factor activity within at least 50%,80%, or 90% when compared to the closest related naturally occurringfamily member. An Oct polypeptide may also comprise at least theDNA-binding domain of the naturally occurring family member and mayfurther comprise a transcriptional activation domain. Exemplary Octpolypeptides include Oct-1, Oct-2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9,and Oct-11. Oct3/4 (referred to herein as “Oct4”) and contain a POUdomain, a 150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2,and uric-86. (See, Ryan, A. K. & Rosenfeld, M. G., Genes Dev.11:1207-1225 (1997), incorporated herein by reference in its entirety.)In some embodiments, variants have at least 85%, 90%, or 95% amino acidsequence identity across their whole sequence compared to a naturallyoccurring Oct polypeptide family member such as those listed above or inGenbank accession number NP-002692.2 (human Oct4) or NP-038661.1 (mouseOct4). Oct polypeptides (e.g., Oct3/4 or Oct 4) can be from human,mouse, rat, bovine, porcine, or other animals. In some embodiments, thesame species of protein will be used with the species of cells beingmanipulated. The Oct polypeptide(s) can be a pluripotency factor thatcan help induce multipotency in non-pluripotent cells.

A “Klf polypeptide” refers to any of the naturally-occurring members ofthe family of Krüppel-like factors (Klfs), zinc-finger proteins thatcontain amino acid sequences similar to those of the Drosophilaembryonic pattern regulator Krüppel, or variants of thenaturally-occurring members that maintain transcription factor activitysimilar (within at least 50%, 80%, or 90% activity) compared to theclosest related naturally occurring family member, or polypeptidescomprising at least the DNA-binding domain of the naturally occurringfamily member, and can further comprise a transcriptional activationdomain. (See, Dang, D. T., Pevsner, J. & Yang, V. W., Cell Biol.32:1103-1121 (2000), incorporated by reference herein in its entirety.)Exemplary Klf family members including, Klf1, Klf2, Klf3, Klf-4, Klf5,Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16,and Klf17. Klf2 and Klf-4 were found to be factors capable of generatingiPS cells in mice, and related genes Klf1 and Klf5 did as well, althoughwith reduced efficiency. (See, Nakagawa, et al., Nature Biotechnology26:101-106 (2007), incorporated by reference herein in its entirety.) Insome embodiments, variants have at least 85%, 90%, or 95% amino acidsequence identity across their whole sequence compared to a naturallyoccurring Klf polypeptide family member such as to those listed above orsuch as listed in Genbank accession number CAX16088 (mouse Klf4) orCAX14962 (human Klf4). Klf polypeptides (e.g., Klf1, Klf4, and Klf5) canbe from human, mouse, rat, bovine, porcine, or other animals. Generally,the same species of protein will be used with the species of cells beingmanipulated. The Klf polypeptide(s) can be a pluripotency factor. Theexpression of the Klf4 gene or polypeptide can help induce multipotencyin a starting cell or a population of starting cells.

A “Myc polypeptide” refers to any of the naturally-occurring members ofthe Myc family. (See, e.g., Adhikary, S. & Eilers, M., Nat. Rev. Mol.Cell Biol. 6:635-645 (2005), incorporated by reference herein in itsentirety.) It also includes variants that maintain similar transcriptionfactor activity when compared to the closest related naturally occurringfamily member (i.e. within at least 50%, 80%, or 90% activity). Itfurther includes polypeptides comprising at least the DNA-binding domainof a naturally occurring family member, and can further comprise atranscriptional activation domain. Exemplary Myc polypeptides include,e.g., c-Myc, N-Myc and L-Myc. In some embodiments, variants have atleast 85%, 90%, or 95% amino acid sequence identity across their wholesequence compared to a naturally occurring Myc polypeptide familymember, such as to those listed above or such as listed in Genbankaccession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc)can be from human, mouse, rat, bovine, porcine, or other animals.Generally, the same species of protein will be used with the species ofcells being manipulated. The Myc polypeptide(s) can be a pluripotencyfactor.

A “Sox polypeptide” refers to any of the naturally-occurring members ofthe SRY-related HMG-box (Sox) transcription factors, characterized bythe presence of the high-mobility group (HMG) domain, or variantsthereof that maintain similar transcription factor activity whencompared to the closest related naturally occurring family member (i.e.within at least 50%, 80%, or 90% activity). It also includespolypeptides comprising at least the DNA-binding domain of the naturallyoccurring family member and can further comprise a transcriptionalactivation domain. (See, e.g., Dang, D. T. et al., Int. J. Biochem. CellBiol. 32:1103-1121 (2000), incorporated by reference herein in itsentirety.) Exemplary Sox polypeptides include, e.g., Sox1, Sox-2, Sox3,Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14,Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 has been shown to yield iPScells with a similar efficiency as Sox2, and genes Sox3, Sox15, andSox18 have also been shown to generate iPS cells, although with somewhatless efficiency than Sox2. (See, Nakagawa, et al., Nature Biotechnology26:101-106 (2007), incorporated by reference herein in its entirety.) Insome embodiments, variants have at least 85%, 90%, or 95% amino acidsequence identity across their whole sequence compared to a naturallyoccurring Sox polypeptide family member such as to those listed above orsuch as listed in Genbank accession number CAA83435 (human Sox2). Soxpolypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can be fromhuman, mouse, rat, bovine, porcine, or other animals. Generally, thesame species of protein will be used with the species of cells beingmanipulated. The Sox polypeptide(s) can be a pluripotency factor. Asdiscussed herein, SOX2 proteins find particular use in the generation ofiPSCs.

By “differentiated ABO− cell” e.g., differentiated PSCO− or HIPO cellsherein is meant pluripotent cells that are blood group O, Rh factor—andmay have been engineered to possess hypoimmunogenicity (e.g. by theknock out of B2M and CIITA and the knock in of CD47) and then aredifferentiated into a cell type for ultimate transplantation intosubjects. Thus, for example HIPO− cells can be differentiated intohepatocytes (“dHIPO− hepatocytes”), into beta-like pancreatic cells orislet organoids (“dHIPO− beta cells”), into endothelial cells (“dHIPO−endothelial cells”), etc.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refers to two or more sequences orsubsequences that have a specified percentage of nucleotides or aminoacid residues that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the sequence comparisonalgorithms described below (e.g., BLASTP and BLASTN or other algorithmsavailable to persons of skill) or by visual inspection. Depending on theapplication, the percent “identity” can exist over a region of thesequence being compared, e.g., over a functional domain, or,alternatively, exist over the full length of the two sequences to becompared. For sequence comparison, typically one sequence acts as areference sequence to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

“Inhibitors,” “activators,” and “modulators” affect a function orexpression of a biologically-relevant molecule. The term “modulator”includes both inhibitors and activators. They may be identified using invitro and in vivo assays for expression or activity of a targetmolecule.

“Inhibitors” are agents that, e.g., inhibit expression or bind to targetmolecules or proteins. They may partially or totally block stimulationor have protease inhibitor activity. They may reduce, decrease, prevent,or delay activation, including inactivation, desensitizion, or downregulation of the activity of the described target protein. Modulatorsmay be antagonists of the target molecule or protein.

“Activators” are agents that, e.g., induce or activate the function orexpression of a target molecule or protein. They may bind to, stimulate,increase, open, activate, or facilitate the target molecule activity.Activators may be agonists of the target molecule or protein.

“Homologs” are bioactive molecules that are similar to a referencemolecule at the nucleotide sequence, peptide sequence, functional, orstructural level. Homologs may include sequence derivatives that share acertain percent identity with the reference sequence. Thus, in oneembodiment, homologous or derivative sequences share at least a 70percent sequence identity. In a specific embodiment, homologous orderivative sequences share at least an 80 or 85 percent sequenceidentity. In a specific embodiment, homologous or derivative sequencesshare at least a 90 percent sequence identity. In a specific embodiment,homologous or derivative sequences share at least a 95 percent sequenceidentity. In a more specific embodiment, homologous or derivativesequences share at least an 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity.Homologous or derivative nucleic acid sequences may also be defined bytheir ability to remain bound to a reference nucleic acid sequence underhigh stringency hybridization conditions. Homologs having a structuralor functional similarity to a reference molecule may be chemicalderivatives of the reference molecule. Methods of detecting, generating,and screening for structural and functional homologs as well asderivatives are known in the art.

“Hybridization” generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel et al,Current Protocols in Molecular Biology, Wiley Interscience Publishers(1995), incorporated by reference herein in its entirety.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.

“Stringent conditions” or “high stringency conditions”, as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 Mm sodium phosphate buffer at Ph 6.5with 750 Mm sodium chloride, 75 Mm sodium citrate at 42° C.; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 Mm sodium phosphate (Ph 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μl/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

As used herein the term “modification” refers to an alteration thatphysically differentiates the modified molecule from the parentmolecule. In one embodiment, an amino acid change in a CD47, HSVtk,EC-CD, or iCasp9 variant polypeptide prepared according to the methodsdescribed herein differentiates it from the corresponding parent thathas not been modified according to the methods described herein, such aswild-type proteins, a naturally occurring mutant proteins or anotherengineered protein that does not include the modifications of suchvariant polypeptide. In another embodiment, a variant polypeptideincludes one or more modifications that differentiates the function ofthe variant polypeptide from the unmodified polypeptide. For example, anamino acid change in a variant polypeptide affects its receptor bindingprofile. In other embodiments, a variant polypeptide comprisessubstitution, deletion, or insertion modifications, or combinationsthereof. In another embodiment, a variant polypeptide includes one ormore modifications that increases its affinity for a receptor comparedto the affinity of the unmodified polypeptide.

In one embodiment, a variant polypeptide includes one or moresubstitutions, insertions, or deletions relative to a correspondingnative or parent sequence. In certain embodiments, a variant polypeptideincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 to 40, 41 to 50, or51 or more modifications.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical value/range, it modifies that value/range by extending theboundaries above and below the numerical value(s) set forth. In general,the term “about” is used herein to modify a numerical value(s) above andbelow the stated value(s) by a variance of 20%.

“Modulated,” as used herein with respect to protein expression, meansthat protein expression level is increased or decreased relative to thecorresponding wild type level for that protein, e.g., the expression ofa protein in a modified pluripotent cell is modulated if it is increasedor decreased relative to the protein expression level of the unmodified(wild type) pluripotent cell.

By “episomal vector” herein is meant a genetic vector that can exist andreplicate autonomously in the cytoplasm of a cell; e.g. it is notintegrated into the genomic DNA of the host cell. A number of episomalvectors are known in the art and described below.

As used herein, the terms “evade rejection,” “escape rejection,” “avoidrejection,” and similar terms are used interchangeably to refer togenetically or otherwise modified cells according to the invention thatare less susceptible to rejection when transplanted into a subject whencompared with corresponding cells that are not genetically modifiedaccording to the invention. In some embodiments, the geneticallymodified cells according to the invention are less susceptible torejection when transplanted into a subject when compared withcorresponding cells that are ABO blood group or Rh factor mismatched tothe subject.

VII. CELLS OF THE INVENTION

The invention provides compositions and methodologies for generatingblood type O− pluripotent (PSCO−) cells. In some aspects of theinvention, the cells will be O− induced pluripotent stem (iPSCO−) cells,O− embryonic stem (ESCO−) cells, hypoimmunogenic pluripotent O− (HIPO−)cells, or cells derived or differentiated therefrom. In other aspects,the unmodified cell type is O−. In other aspects, the cells are modifiedenzymatically or genetically to the O− Rh− blood type.

A. Methodologies for Genetic Alterations

The invention includes methods of modifying nucleic acid sequenceswithin cells or in cell-free conditions to generate both pluripotentcells and HIP cells. Exemplary technologies include homologousrecombination, knock-in, ZFNs (zinc finger nucleases), TALENs(transcription activator-like effector nucleases), CRISPR/Cas9, andother site-specific nuclease technologies. These techniques enabledouble-strand DNA breaks at desired locus sites. These controlleddouble-strand breaks promote homologous recombination at the specificlocus sites. This process focuses on targeting specific sequences ofnucleic acid molecules, such as chromosomes, with endonucleases thatrecognize and bind to the sequences and induce a double-stranded breakin the nucleic acid molecule. The double-strand break is repaired eitherby an error-prone non-homologous end-joining (NHEJ) or by homologousrecombination (HR).

As will be appreciated by those in the art, a number of differenttechniques can be used to engineer the pluripotent cells of theinvention, as well as the engineering of the PSCs or iPSCs to becomeblood type O, Rh negative and optionally hypo-immunogenic as outlinedherein.

In general, these techniques can be used individually or in combination.For example, in the generation of the HIP cells, CRISPR may be used toreduce the expression of active B2M and/or CIITA protein in theengineered cells, with viral techniques (e.g. lentivirus) to knock inthe CD47 functionality. Also, as will be appreciated by those in theart, although one embodiment sequentially utilizes a CRISPR step toknock out B2M, followed by a CRISPR step to knock out CIITA with a finalstep of a lentivirus to knock in the CD47 functionality, these genes canbe manipulated in different orders using different technologies.

As is discussed more fully below, transient expression of reprogramminggenes is generally done to generate/induce pluripotent stem cells.

a. CRISPR Technologies

In one embodiment, the cells are manipulated using CRISPR/Castechnologies as is known in the art. CRISPR can be used to generate thestarting PSCO−, iPSCO−, or ESCO− or to generate the HIPO− cells from thePSCO-s, iPSCO-s or ESCO-s. There are a large number of techniques basedon CRISPR, see for example Doudna and Charpentier, Sciencedoi:10.1126/science.1258096, hereby incorporated by reference. CRISPRtechniques and kits are sold commercially.

b. TALEN Technologies

In some embodiments, the HIP cells of the invention are made usingTranscription Activator-Like Effector Nucleases (TALEN) methodologies.TALEN are restriction enzymes combined with a nuclease that can beengineered to bind to and cut practically any desired DNA sequence.TALEN kits are sold commercially.

c. Zinc Finger Technologies

In one embodiment, the cells are manipulated using Zn finger nucleasetechnologies. Zn finger nucleases are artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target specific desiredDNA sequences and this enables zinc-finger nucleases to target uniquesequences within complex genomes. By taking advantage of endogenous DNArepair machinery, these reagents can be used to precisely alter thegenomes of higher organisms, similar to CRISPR and TALENs.

d. Viral Based Technologies

There are a wide variety of viral techniques that can be used togenerate the HIP cells of the invention (as well as for the originalgeneration of the iPCSs), including, but not limited to, the use ofretroviral vectors, lentiviral vectors, adenovirus vectors and Sendaiviral vectors. Episomal vectors used in the generation of iPSCs aredescribed below.

e. Down regulation of genes using interfering RNA

In other embodiments, genes that encode proteins used in HLA moleculesare down regulated by RNAi technologies. RNA interference (RNAi) is aprocess where RNA molecules inhibit gene expression often by causingspecific mRNA molecules to degrade. Two types of RNA molecules—microRNA(miRNA) and small interfering RNA (siRNA)—are central to RNAinterference. They bind to the target mRNA molecules and either increaseor decrease their activity. RNAi helps cells defend against parasiticnucleic acids such as those from viruses and transposons. RNAi alsoinfluences development.

sdRNA molecules are a class of asymmetric siRNAs comprising a guide(antisense) strand of 19-21 bases. They contain a 5′ phosphate, 2′Ome or2′F modified pyrimidines, and six phosphotioates at the 3′ positions.They also contain a sense strand containing 3′ conjugated sterolmoieties, 2 phospotioates at the 3′ position, and 2′Ome modifiedpyrimidines. Both strands contain 2′ Ome purines with continuousstretches of unmodified purines not exceeding a length of 3. sdRNA isdisclosed in U.S. Pat. No. 8,796,443, incorporated herein by referencein its entirety.

For all of these technologies, well known recombinant techniques areused, to generate recombinant nucleic acids as outlined herein. Incertain embodiments, the recombinant nucleic acids (either encoding adesired polypeptide, e.g. CD47, or disruption sequences) may be operablylinked to one or more regulatory nucleotide sequences in an expressionconstruct. Regulatory nucleotide sequences will generally be appropriatefor the host cell and subject to be treated. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, the one or moreregulatory nucleotide sequences may include, but are not limited to,promoter sequences, leader or signal sequences, ribosomal binding sites,transcriptional start and termination sequences, translational start andtermination sequences, and enhancer or activator sequences. Constitutiveor inducible promoters as known in the art are also contemplated. Thepromoters may be either naturally occurring promoters, or hybridpromoters that combine elements of more than one promoter. An expressionconstruct may be present in a cell on an episome, such as a plasmid, orthe expression construct may be inserted in a chromosome. In a specificembodiment, the expression vector includes a selectable marker gene toallow the selection of transformed host cells. Certain embodimentsinclude an expression vector comprising a nucleotide sequence encoding avariant polypeptide operably linked to at least one regulatory sequence.Regulatory sequences for use herein include promoters, enhancers, andother expression control elements. In certain embodiments, an expressionvector is designed for the choice of the host cell to be transformed,the particular variant polypeptide desired to be expressed, the vector'scopy number, the ability to control that copy number, or the expressionof any other protein encoded by the vector, such as antibiotic markers.

Examples of suitable mammalian promoters include, for example, promotersfrom the following genes: ubiquitin/S27a promoter of the hamster (WO97/15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirusmajor late promoter, mouse metallothionein-I promoter, the long terminalrepeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor viruspromoter (MMTV), Moloney murine leukemia virus Long Terminal repeatregion, and the early promoter of human Cytomegalovirus (CMV). Examplesof other heterologous mammalian promoters are the actin, immunoglobulinor heat shock promoter(s).

In additional embodiments, promoters for use in mammalian host cells canbe obtained from the genomes of viruses such as polyoma virus, fowlpoxvirus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40). In further embodiments, heterologousmammalian promoters are used. Examples include the actin promoter, animmunoglobulin promoter, and heat-shock promoters. The early and latepromoters of SV40 are conveniently obtained as an SV40 restrictionfragment which also contains the SV40 viral origin of replication. Fierset al., Nature 273: 113-120 (1978). The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment. Greenaway, P. J. et al., Gene 18: 355-360 (1982).The foregoing references are incorporated by reference in theirentirety.

B. Generation of Pluripotent Cells

The invention provides methods of producing non-immunogenic pluripotentcells from pluripotent cells. Thus, the first step is to provide thepluripotent stem cells.

The generation of mouse and human pluripotent stem cells (generallyreferred to as iPSCs; miPSCs for murine cells or hiPSCs for human cells)is generally known in the art. As will be appreciated by those in theart, there are a variety of different methods for the generation ofiPCSs. The original induction was done from mouse embryonic or adultfibroblasts using the viral introduction of four transcription factors,Oct3/4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell126:663-676 (2006), hereby incorporated by reference in its entirety andspecifically for the techniques outlined therein. Since then, a numberof methods have been developed; see Seki et al., World J. Stem Cells7(1):116-125 (2015) for a review, and Lakshmipathy and Vermuri, editors,Methods in Molecular Biology: Pluripotent Stem Cells, Methods andProtocols, Springer 2013, both of which are hereby expresslyincorporated by reference in their entirety, and in particular for themethods for generating hiPSCs (see for example Chapter 3 of the latterreference).

Generally, iPSCs are generated by the transient expression of one ormore “reprogramming factors” in the host cell, usually introduced usingepisomal vectors. Under these conditions, small amounts of the cells areinduced to become iPSCs (in general, the efficiency of this step is low,as no selection markers are used). Once the cells are “reprogrammed”,and become pluripotent, they lose the episomal vector(s) and produce thefactors using the endogeneous genes. This loss of the episomal vector(s)results in cells that are called “zero footprint” cells. This isdesirable as the fewer genetic modifications (particularly in the genomeof the host cell), the better. Thus, it is preferred that the resultinghiPSCs have no permanent genetic modifications.

As is also appreciated by those of skill in the art, the number ofreprogramming factors that can be used or are used can vary. Commonly,when fewer reprogramming factors are used, the efficiency of thetransformation of the cells to a pluripotent state goes down, as well asthe “pluripotency”, e.g. fewer reprogramming factors may result in cellsthat are not fully pluripotent but may only be able to differentiateinto fewer cell types.

In some embodiments, a single reprogramming factor, OCT4, is used. Inother embodiments, two reprogramming factors, OCT4 and KLF4, are used.In other embodiments, three reprogramming factors, OCT4, KLF4 and SOX2,are used. In other embodiments, four reprogramming factors, OCT4, KLF4,SOX2 and c-Myc, are used. In other embodiments, 5, 6 or 7 reprogrammingfactors can be used selected from SOKMNLT; SOX2, OCT4 (POU5F1), KLF4,MYC, NANOG, LIN28, and SV40L T antigen.

In general, these reprogramming factor genes are provided on episomalvectors such as are known in the art and commercially available. Forexample, ThermoFisher/Invitrogen sell a sendai virus reprogramming kitfor zero footprint generation of hiPSCs, see catalog number A34546.ThermoFisher also sells EBNA-based systems as well, see catalog numberA14703.

In addition, there are a number of commercially available hiPSC lines;see, e.g., the Gibco® Episomal hiPSC line, K18945, which is a zerofootprint, viral-integration-free human iPSC cell line (see alsoBurridge et al, 2011, supra).

In general, as is known in the art, iPSCs are made from non-pluripotentcells such as CD34+ cord blood cells, fibroblasts, etc., by transientlyexpressing the reprogramming factors as described herein.

For example, successful iPSCs were also generated using only Oct3/4,Sox2 and Klf4, while omitting the C-Myc, although with reducedreprogramming efficiency.

In general, iPSCs are characterized by the expression of certain factorsthat include KLF4, Nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc and TCL1. Newor increased expression of these factors for purposes of the inventionmay be via induction or modulation of an endogenous locus or fromexpression from a transgene.

For example, murine iPSCs can be generated using the methods of Dieckeet al, Sci Rep. 2015, Jan. 28; 5:8081 (doi:10.1038/srep08081), herebyincorporated by reference in its entirety and specifically for themethods and reagents for the generation of the miPSCs. See also, e.g.,Burridge et al., PLoS One, 2011 6(4):18293, hereby incorporated byreference in its entirety and specifically for the methods outlinedtherein.

In some cases, the pluripotency of the cells is measured or confirmed asoutlined herein, for example by assaying for generally accepted markersto indicate plurpotency such as, e.g., Nanog, Oct 4, Sox2, Esrrb, Tbx3,and Tc11. Alternatively, the pluripotency of the cells can be measuredor confirmed by conducting differentiation reactions as outlined in theExamples.

C. Generation of Pluripotent O− Cells

In some aspects of the invention, the iPSC cells generated as discussedabove will already be blood type O, Rh factor negative cells because theprocess will have started with pluripotent cells having an O− bloodtype.

In other embodiments of the invention, the iPSCs can be made blood typeO by enzymatic conversion of A and B antigens. In preferred aspects, theB antigen is converted to 0 using an enzyme. In more preferred aspects,the enzyme is an α-galactosidase. This enzyme eliminates the terminalgalactose residue of the B antigen. Other aspects of the inventioninvolve the enzymatic conversion of A antigen to O. In preferredaspects, the A antigen is converted to O using ancc-N-acetylgalactosaminidase. Enzymatic conversion is discussed, e.g.,in Olsson et al., Transfusion Clinique et Biologique 11:33-39 (2004);U.S. Pat. Nos. 4,427,777, 5,606,042, 5,633,130, 5,731,426, 6,184,017,4,609,627, and 5,606,042; and Intl Pub. No. WO9923210, each of which areincorporated by reference herein in their entirety.

Other embodiments of the invention involve genetically modifying iPSCsto blood type O by engineering the cells to knock out Exon 7 of the ABOgene (NCBI Gene ID: 80908). Any knockout methodology known in the art ordescribed herein, such as CRISPR, TALENs, Zn fingers, or homologousrecombination, may be employed.

Other embodiments of the invention involve rendering the blood type Opluripotent cell Rh factor negative by knocking out the C and/or Eantigens of the Rh blood group system (RH), K in the Kell system (KEL),Fya and Fy3 in the Duffy system (FY), Jkb in the Kidd system (JK), or Uand/or S in the MNS blood group system. Alternatively, the cells of theinvention may be made Rh negative by or silencing the SLC14A1 (JK) gene(NCBI Gene ID: 6563). Any knockout methodology known in the art ordescribed herein, such as CRISPR, TALENs, Zn Fingers, or homologousrecombination, may be employed.

D. Generation of Hypoimmunogenic Pluripotent (HIP) Cells

Generating HIP cells from pluripotent cells or HIPO− cells from PSCO−,iPSCO− cells is done with as few as three genetic changes, resulting inminimal disruption of cellular activity but conferring immunosilencingto the cells.

The first two genetic changes involve a reduction or elimination in theprotein activity of MHC I and II (HLA I and II when the cells arehuman). This can be done by altering genes encoding their component. Inone embodiment, the coding region or regulatory sequences of the geneare disrupted using CRISPR. In another embodiment, gene translation isreduced using interfering RNA technologies. The third change involvesincreasing expression of a gene that regulates susceptibility tomacrophage phagocytosis, such as CD47. This can be effected by using a“knock in” of a gene using viral or transgene technologies.

In some cases, where CRISPR is being used for the genetic modifications,hiPSC or hiPSCO− cells that contain a Cas9 construct that enable highefficiency editing of the cell line can be used; see, e.g., the HumanEpisomal Cas9 iPSC cell line, A33124, from Life Technologies.

1. HLA-I Reduction

The HIPO− cells of the invention include a reduction in MHC I function(HLA I when the cells are derived from human cells).

As will be appreciated by those in the art, the reduction in functioncan be accomplished in a number of ways, including removing nucleic acidsequences from a gene, interrupting the sequence with other sequences,or altering the regulatory components of the nucleic acid. For example,all or part of a coding region of the gene of interest can be removed orreplaced with “nonsense” sequences, frameshift mutations can be made,all or part of a regulatory sequence such as a promoter can be removedor replaced, translation initiation sequences can be removed orreplaced, etc.

As will be appreciated by those in the art, the successful reduction ofthe MHC I function (HLA I when the cells are derived from human cells)in the pluripotent cells can be measured using techniques known in theart and as described below; for example, FACS techniques using labeledantibodies that bind the HLA complex; for example, using commerciallyavailable HLA-A, B, or C antibodies that bind to the alpha chain of thehuman major histocompatibility HLA Class I antigens.

a. B2M Alteration

In one embodiment, the reduction in HLA-I activity is accomplished byreducing β-2 microglobulin expression. This may be achieved bydisrupting the expression of the β-2 microglobulin gene in thepluripotent stem cell, the human sequence of which is disclosed herein.This alteration is generally referred to herein as a gene “knock out”,and in the HIP cells of the invention it is done on both alleles in thehost cell. Generally, the techniques to do both disruptions are thesame.

A particularly useful embodiment uses CRISPR technology to disrupt thegene. In some cases, CRISPR technology is used to introduce smalldeletions or insertions into the coding region of the gene such that nofunctional protein is produced. Often, the result of frameshiftmutations result in the generation of stop codons and truncated,non-functional proteins are made.

Accordingly, a useful technique is to use CRISPR sequences designed totarget the coding sequence of the B2M gene in mouse or the B2M gene inhuman. After gene editing, the transfected iPSC cultures are dissociatedto single cells. Single cells are expanded to full-size colonies andtested for CRISPR edits by screening for the presence of aberrantsequences from the CRISPR cleavage site. Clones with deletions in bothalleles are picked. Such clones do not express B2M as demonstrated byPCR and do not express HLA-I as demonstrated by FACS analysis (seeexamples 1 and 6, for example).

Assays to test whether the B2M gene has been inactivated are known anddescribed herein. In one embodiment, the assay is a Western blot of celllysates probed with antibodies to the B2M protein. In anotherembodiment, reverse transcriptase polymerase chain reactions (rt-PCR)confirms the presence of the inactivating alteration.

In addition, the cells can be tested to confirm that the HLA I complexis not expressed on the cell surface. This may be assayed by FACSanalysis using antibodies to one or more HLA cell surface components asdiscussed above.

2. HLA-II Reduction

In addition to a reduction in HLA I, the HIPO− cells of the inventionalso lack MHC II function (HLA II when the cells are derived from humancells).

As will be appreciated by those in the art, the reduction in functioncan be accomplished in a number of ways, including removing nucleic acidsequences from a gene, adding nucleic acid sequences to a gene,disrupting the reading frame, interrupting the sequence with othersequences, or altering the regulatory components of the nucleic acid. Inone embodiment, all or part of a coding region of the gene of interestcan be removed or replaced with “nonsense” sequences. In anotherembodiment, regulatory sequences such as a promoter can be removed orreplaced, translation initiation sequences can be removed or replaced,etc.

The successful reduction of the MHC II function (HLA II when the cellsare derived from human cells) in the pluripotent cells or theirderivatives can be measured using techniques known in the art such asWestern blotting using antibodies to the protein, FACS techniques,rt-PCR techniques, etc.

a. a ITA Alteration

In one embodiment, the reduction in HLA-II activity is done bydisrupting the expression of the CIITA gene in the pluripotent stemcell, the human sequence of which is shown herein. This alteration isgenerally referred to herein as a gene “knock out”, and in the HIPO−cells of the invention it is done on both alleles in the host cell.

Assays to test whether the CIITA gene has been inactivated are known anddescribed herein. In one embodiment, the assay is a Western blot of celllysates probed with antibodies to the CIITA protein. In anotherembodiment, reverse transcriptase polymerase chain reactions (rt-PCR)confirms the presence of the inactivating alteration.

In addition, the cells can be tested to confirm that the HLA II complexis not expressed on the cell surface. Again, this assay is done as isknown in the art and generally is done using either Western Blots orFACS analysis based on commercial antibodies that bind to human HLAClass II HLA-DR, DP and most DQ antigens as outlined below.

A particularly useful embodiment uses CRISPR technology to disrupt theCIITA gene. CRISPRs are designed to target the coding sequence of theCIITA gene, an essential transcription factor for all MHC II molecules.After gene editing, the transfected iPSC cultures are dissociated intosingle cells. They are expanded to full-size colonies and tested forsuccessful CRISPR editing by screening for the presence of an aberrantsequence from the CRISPR cleavage site. Clones with deletions do notexpress CIITA as determined by PCR and do not express MHC II/HLA-II asdetermined by FACS analysis.

3. Phagocytosis Reduction

In addition to the reduction of HLA I and II (or MHC I and II),generally using B2M and CIITA knock-outs, the HIPO− cells of theinvention have a reduced susceptibility to macrophage phagocytosis andNK cell killing. The resulting HIPO− cells “escape” the immunemacrophage and innate pathways due to one or more CD47 transgenes.

a. CD47 Increase

In some embodiments, reduced macrophage phagocytosis and NK cell killingsusceptibility results from increased CD47 on the HIPO− cell surface.This is done in several ways as will be appreciated by those in the artusing “knock in” or transgenic technologies. In some cases, increasedCD47 expression results from one or more CD47 transgenes.

Accordingly, in some embodiments, one or more copies of a CD47 gene isadded to the HIPO− cells under control of an inducible or constitutivepromoter, with the latter being preferred. In some embodiments, alentiviral construct is employed as described herein or known in theart. CD47 genes may integrate into the genome of the host cell under thecontrol of a suitable promoter as is known in the art.

The HIPO− cell lines can be generated from B2M−/− CIITA−/− iPSCs. Cellscontaining lentivirus vectors expressing CD47 may be selected using aBlasticidin marker. The CD47 gene sequence is synthesized and the DNA iscloned into the plasmid Lentivirus pLenti6/V5 with a blasticidinresistance (Thermo Fisher Scientific, Waltham, Mass.)

In some embodiments, the expression of the CD47 gene can be increased byaltering the regulatory sequences of the endogenous CD47 gene, forexample, by exchanging the endogenous promoter for a constitutivepromoter or for a different inducible promoter. This can generally bedone using known techniques such as CRISPR.

Once altered, the presence of sufficient CD47 expression can be assayedusing known techniques such as those described in the Examples, such asWestern blots, ELISA assays or FACS assays using anti-CD47 antibodies.In general, “sufficiency” in this context means an increase in theexpression of CD47 on the HIPO− cell surface that silences NK cellkilling. The natural expression levels on cells are too low to protectthem from NK cell lysis once their MHC I is removed.

4. Other Modifications

In some embodiments, the hypoimmunogenic cells are generated by editingthe CCR5 and/or CXCR4 genes to reduce or eliminate CCR5 and/or CXCR4protein surface expression, by using genome editing tools such as forexample CRISPR or TALEN, as described in WO2016/073955, incorporated byreference herein in its entirety, and specifically for the relatedtechniques outlined therein. In some embodiments, the hypoimmunogeniccells are generated by editing the CCR5 and/or CXCR4 genes in additionto the B2M gene to reduce or eliminate MHC class 1 surface expression asdescribed in WO2016/073955, incorporated by reference herein in itsentirety, and specifically for the related techniques outlined therein.

In some embodiments, the hypoimmunogenic cells are generated by editingthe NLRC5, CIITA, and/or B2M genes to reduce or eliminate surfaceexpression and/or activity of the NLRC5, CIITA, and/or B2M proteins, byuse of genome editing tools such as for example CRISPR or TALEN, asdescribed in WO2016/183041, incorporated by reference herein in itsentirety, and specifically for the related techniques outlined therein.In some embodiments, the hypoimmunogenic pluripotent cells are generatedby modulating expression of one or more tolerogenic factor genes such asHLA-A, HLA-B, HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, Cl-inhibitor,and IL-35 as described in WO2016/183041, incorporated by referenceherein in its entirety, and specifically for the related techniquesoutlined therein. In some embodiments, the hypoimmunogenic cells aregenerating by modulating expression of one or more genes such as RFX-5,RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, and/or IRF-1 as described inWO2016/183041, incorporated by reference herein in its entirety, andspecifically for the related techniques outlined therein. In additionalembodiments, further modifications to the cells include modulatingexpression of one or more genes such as OX40, GITR, 4-1BB, CD28, B7-1,B7-2, ICOS, CD27, HVEM, SLAM, CD226, PD1, CTL4, LAG3, TIGIT, TIM3,CD160, BTLA, CD244, CD30, TLT, VISTA, B7-H3, PD-L2, LFA-1, CD2, CD58,ICAM-3, TCRA, TCRB, FOXP3, HELIOS, ST2, PCSK9, CCR5, and/or APOC3 asdescribed in WO2016/183041, incorporated by reference herein in itsentirety, and specifically for the related techniques outlined therein.

In some embodiments, the hypoimmunogenic cells are generated by theintroduction of one or more transgenes such as PDL-1, HLA-G, CD47,CD200, FASLG, CLC21, MFGE8, and/or SERPIN B9, or a gene encoding abiologic that acts as an agonist of PDL-1, HLA-G, CD47, CD200, FASLG,CLC21, MFGE8, and/or SERPIN 9, as described in WO2018/227286,incorporated by reference herein in its entirety, and specifically forthe related techniques outlined therein. In some embodiments, thehypoimmunogenic cell is generated by further introduction of one or moretransgenes such as TGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22,TNFRSF23, TNFRS10, DAD1, and/or IFNγR1 d39, or a gene encoding abiologic that acts as an agonist of TGFβ, CD73, CD39, LAG3, IL1R2,ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1, and/or IFNγR1 d39, asdescribed in WO2018/227286, incorporated by reference herein in itsentirety, and specifically for the related techniques outlined therein.

5. Suicide Genes

In some embodiments, the invention provides HIPO− cells that comprise a“suicide gene” or “suicide switch”. These are incorporated to functionas a “safety switch” that can cause the death of the hypoimmunogenicpluripotent cells should they grow and divide in an undesired manner.The “suicide gene” ablation approach includes a suicide gene in a genetransfer vector encoding a protein that results in cell killing onlywhen activated by a specific compound. A suicide gene may encode anenzyme that selectively converts a nontoxic compound into highly toxicmetabolites. The result is specifically eliminating cells expressing theenzyme. In some embodiments, the suicide gene is the herpesvirusthymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In otherembodiments, the suicide gene is the Escherichia coli cytosine deaminase(EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al.,Mol. Therap. 20(10):1932-1943 (2012), Xu et al., Cell Res. 8:73-8(1998), both incorporated herein by reference in their entirety.)

In other embodiments, the suicide gene is an inducible Caspase protein.An inducible Caspase protein comprises at least a portion of a Caspaseprotein capable of inducing apoptosis. In one embodiment, the portion ofthe Caspase protein is exemplified in SEQ ID NO:6. In preferredembodiments, the inducible Caspase protein is iCasp9. It comprises thesequence of the human FK506-binding protein, FKBP12, with an F36Vmutation, connected through a series of amino acids to the gene encodinghuman caspase 9. FKBP12-F36V binds with high affinity to asmall-molecule dimerizing agent, AP1903. Thus, the suicide function ofiCasp9 in the instant invention is triggered by the administration of achemical inducer of dimerization (CID). In some embodiments, the CID isthe small molecule drug AP1903. Dimerization causes the rapid inductionof apoptosis. (See WO2011146862; Stasi et al, N. Engl. J. Med 365; 18(2011); Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007),each of which are incorporated by reference herein in their entirety.)

6. Assays for HIPO− Phenotypes and Retention of Pluripotency

Once the HIPO− cells have been generated, they may be assayed for theirhypoimmunogenicity and/or retention of pluripotency as is generallydescribed herein and in WO 2018/132783, incorporated herein byreference.

For example, hypoimmunogenicity may be assayed using a number oftechniques. These techniques include transplantation into allogeneichosts and monitoring for HIPO− cell growth (e.g. teratomas) that escapethe host immune system. HIPO− derivatives are transduced to expressluciferase and can then be followed using bioluminescence imaging.Similarly, the T cell and/or B cell response of the host animal to theHIP cells can be tested to confirm that the HIP cells do not cause animmune reaction in the host animal. T cell function can be assessed byElispot, Elisa, FACS, PCR, or mass cytometry (CYTOF). B cell response orantibody response can be assessed using FACS or luminex. Additionally oralternatively, the cells may be assayed for their ability to avoidinnate immune responses, e.g. NK cell killing. K cell lytolytic activitycan be assessed in vitro or in vivo.

Similarly, the retention of pluripotency can be tested in a number ofways. In one embodiment, pluripotency is assayed by the expression ofcertain pluripotency-specific factors as generally described herein andin WO 2018/0132783. Additionally or alternatively, the HIPO− cells maybe differentiated into one or more cell types as an indication ofpluripotency.

E. Embodiments of the Invention

The iPSCO−, ESCO−, and HIPO− cells, or derivatives thereof, of theinvention may be used to treat, for example, Type 1 diabetes, cardiacdiseases, neurological diseases, cancer, blindness, vascular diseases,and other diseases/disorders that respond to regenerative medicinetherapies. In particular, the invention contemplates using the HIPO−cells for differentiation into any cell type. Thus, provided herein areiPSCO−, ESCO−, and HIPO− cells, or derivatives thereof, that exhibitpluripotency but do not result in a host immune response whentransplanted into an allogeneic host such as a human patient, either asthe iPSCO−, ESCO−, and HIPO− cells, or as the differentiated products ofthe those cells.

In one aspect, the present invention provides an isolated iPSCO−, ESCO−,and HIPO− cell, or derivative thereof, comprising a nucleic acidencoding a chimeric antigen receptor (CAR), wherein endogenous β-2microglobulin (B2M) gene activity and endogenous class II transactivator(CIITA) gene activity have been eliminated and CD47 expression has beenincreased. The CAR can comprise an extracellular domain, a transmembranedomain, and an intracellular signaling domain. In some embodiments, theextracellular domain binds to an antigen selected from the groupconsisting of CD19, CD20, CD22, CD38, CD123, CS1, CD171, BCMA, MUC16,ROR1, and WT1. In certain embodiments, the extracellular domaincomprises a single chain variable fragment (scFv). In some embodiments,the transmembrane domain comprises CD3ζ, CD4, CD8α, CD28, 4-1BB, OX40,ICOS, CTLA-4, PD-1, LAG-3, and BTLA. In certain embodiments, theintracellular signaling domain comprises CD3ζ, CD28, 4-1BB, OX40, ICOS,CTLA-4, PD-1, LAG-3, and BTLA.

In certain embodiments, the CAR comprises an anti-CD19 scFv domain, aCD28 transmembrane domain, and a CD3 zeta signaling intracellulardomain. In some embodiments, the CAR comprises an anti-CD19 scFv domain,a CD28 transmembrane domain, a 4-1BB signaling intracellular domain, anda CD3 zeta signaling intracellular domain.

In another aspect of the invention, provided is an isolated O− CAR-T(O-CAR-T) or hypoimmune O− CAR-T (HIPO-CAR-T) cell produced by in vitrodifferentiation of any one of the iPSCO−, ESCO−, and HIPO− cells, orderivatives thereof, described herein. In some embodiments, theHIPO-CAR-T cell is a cytotoxic HIPO− CAR-T cell.

In various embodiments, the in vitro differentiation comprises culturingthe iPSCO−, ESCO−, and HIPO− cell, or derivative thereof, carrying a CARconstruct in a culture media comprising one or more growth factors orcytokines selected from the group consisting of bFGF, EPO, Flt3L, IGF,IL-3, IL-6, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, theculture media further comprises one or more growth factors or cytokinesselected from the group consisting of a BMP activator, a GSK3 inhibitor,a ROCK inhibitor, a TGFβ receptor/ALK inhibitor, and a NOTCH activator.

In particular embodiments, the isolated O-CAR-T or HIPO-CAR-T cellsproduced by in vitro differentiation of any one of the iPSCO−, ESCO−,and HIPO− cell-carrying the CAR-T constructs may be used in thetreatment of cancer.

In another aspect of the invention, provided is a method of treating apatient with cancer by administering a composition comprising atherapeutically effective amount of any of the isolated O-CAR-T orHIPO-CAR-T cells described herein. In some embodiments, the compositionfurther comprises a therapeutically effective carrier.

In some embodiments, the administration step comprises intravenousadministration, subcutaneous administration, intranodal administration,intratumoral administration, intrathecal administration, intrapleuraladministration, and intraperitoneal administration. In certaininstances, the administration further comprises a bolus or by continuousperfusion.

In some embodiments, the cancer is a blood cancer selected from thegroup consisting of leukemia, lymphoma, and myeloma. In variousembodiments, the cancer is a solid tumor cancer or a liquid tumorcancer.

In another aspect, the present invention provides a method of making anyone of the isolated O-CAR-T or HIPO-CAR-T cells described herein. Themethod includes in vitro differentiating of any one of the iPSCO−,ESCO−, or HIPO− cells of the invention wherein in vitro differentiatingcomprises culturing the iPSCO−, ESCO−, or HIPO− cell in a culture mediacomprising one or more growth factors or cytokines selected from thegroup consisting of bFGF, EPO, Flt3L, IGF, IL-2, IL-3, IL-6, IL-7,IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture mediafurther comprises one or more growth factors or cytokines selected fromthe group consisting of a BMP activator, a GSK3 inhibitor, a ROCKinhibitor, a TGFβ receptor/ALK inhibitor, and a NOTCH activator.

In some embodiments, the in vitro differentiating comprises culturingthe iPSCO−, ESCO−, or HIPO-cells on feeder cells. In variousembodiments, the in vitro differentiating comprises culturing insimulated microgravity. In certain instances, the culturing in simulatedmicrogravity is for at least 72 hours.

In some aspects, provided herein is an isolated, engineered hypoimmunecardiac cell (hypoimmunogenic cardiac cell), for example acardiomyocyte, differentiated from an iPSCO−, ESCO−, or HIPO− cell.

Cardiomyocytes were previously thought to lack ABO blood group antigens.Differentiation of an ABO blood group type B human embryonic stem cellline into cardiomyocyte-like cells was observed to result in the loss ofthe B antigen, suggesting that loss of these antigens may occur earlyduring human embryogenesis. See, e.g., Mölne et al., Transplantation.86(10):1407-13 (2008), incorporated by reference herein in its entirety.Other studies also reported that differentiation of induced humanpluripotent stem cells into cardiomyocyte-like cells caused theprogressive loss of the ABO blood group type A antigen in these cells.See, e.g., Säljö et al., Scientific Reports. 13072: 1-14 (2017).Surprisingly, however, the inventors determined that cardiomyocytesexpress ABO blood group antigens that can cause rejection of such cellsto an unmatched recipient.

Accordingly, in some aspects, provided herein is a method of treating apatient suffering from a heart condition or disease. The methodcomprises administering a composition comprising a therapeuticallyeffective amount of a population of any one of the isolated, engineeredO− or hypoimmune O− cardiac cells derived from iPSCO−, ESCO−, or HIPO−cells as described herein. In some embodiments, the composition furthercomprises a therapeutically effective carrier.

In some embodiments, the administration comprises implantation into thepatient's heart tissue, intravenous injection, intraarterial injection,intracoronary injection, intramuscular injection, intraperitonealinjection, intramyocardial injection, trans-endocardial injection,trans-epicardial injection, or infusion.

In some embodiments, the heart condition or disease is selected from thegroup consisting of pediatric cardiomyopathy, age-relatedcardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartumcardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy,myocarditis, myocardial ischemic reperfusion injury, ventriculardysfunction, heart failure, congestive heart failure, coronary arterydisease, end stage heart disease, atherosclerosis, ischemia,hypertension, restenosis, angina pectoris, rheumatic heart, arterialinflammation, or cardiovascular disease.

In some aspects, provided herein is a method of producing a populationof 0-hypoimmune cardiac cells from a population of HIPO− cells by invitro differentiation, wherein endogenous 13-2 microglobulin (B2M) geneactivity and endogenous class II transactivator (CIITA) gene activityhave been eliminated and CD47 expression has been increased as comparedto an unmodified iPSCO− cells. The method comprises: (a) culturing apopulation of HIPO− cells in a culture medium comprising a GSKinhibitor; (b) culturing the population of HIPO− cells in a culturemedium comprising a WNT antagonist to produce a population ofpre-cardiac cells; and (c) culturing the population of pre-cardiac cellsin a culture medium comprising insulin to produce a population of0-hypoimmune cardiac cells. In some embodiments, the GSK inhibitor isCHIR-99021, a derivative thereof, or a variant thereof. In someinstances, the GSK inhibitor is at a concentration ranging from about 2μM to about 10 μM. In some embodiments, the WNT antagonist is IWR1, aderivative thereof, or a variant thereof. In some instances, the WNTantagonist is at a concentration ranging from about 2 μM to about 10 μM.

In some aspects, provided herein is an isolated, engineered O− orO-hypoimmune endothelial cell differentiated from iPSCO−, ESCO−, orHIPO− cells. In other aspects, the isolated, engineered O− orO-hypoimmune endothelial cell is selected from the group consisting of acapillary endothelial cell, vascular endothelial cell, aorticendothelial cell, brain endothelial cell, and renal endothelial cell.

In some aspects, provided herein is a method of treating a patientsuffering from a vascular condition or disease. In some embodiments, themethod comprises administering a composition comprising atherapeutically effective amount of a population of isolated, engineeredO− or O− hypoimmune endothelial cells.

In some embodiments, the method comprises administering a compositioncomprising a therapeutically effective amount of a population of any oneof the isolated, engineered O− or O− hypoimmune endothelial cellsdescribed herein. In some embodiments, the composition further comprisesa therapeutically effective carrier. In some embodiments, theadministration comprises implantation into the patient's heart tissue,intravenous injection, intraarterial injection, intracoronary injection,intramuscular injection, intraperitoneal injection, intramyocardialinjection, trans-endocardial injection, trans-epicardial injection, orinfusion.

In some embodiments, the vascular condition or disease is selected fromthe group consisting of, vascular injury, cardiovascular disease,vascular disease, ischemic disease, myocardial infarction, congestiveheart failure, hypertension, ischemic tissue injury, limb ischemia,stroke, neuropathy, and cerebrovascular disease.

In some aspects, provided herein is a method of producing a populationof O− hypoimmune endothelial cells from a population of HIPO− cells byin vitro differentiation, wherein endogenous β-2 microglobulin (B2M)gene activity and endogenous class II transactivator (CIITA) geneactivity have been eliminated and CD47 expression has been increased inthe HIPO-cells. The method comprises: (a) culturing a population ofHIPO-cells in a first culture medium comprising a GSK inhibitor; (b)culturing the population of HIPO-cells in a second culture mediumcomprising VEGF and bFGF to produce a population of pre-endothelialcells; and (c) culturing the population of pre-endothelial cells in athird culture medium comprising a ROCK inhibitor and an ALK inhibitor toproduce a population of hypoimmune endothelial cells.

In some embodiments, the GSK inhibitor is CHIR-99021, a derivativethereof, or a variant thereof. In some instances, the GSK inhibitor isat a concentration ranging from about 1 μM to about 10 μM. In someembodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or avariant thereof. In some instances, the ROCK inhibitor is at aconcentration ranging from about 1 μM to about 20 μM. In someembodiments, the ALK inhibitor is SB-431542, a derivative thereof, or avariant thereof. In some instances, the ALK inhibitor is at aconcentration ranging from about 0.5 μM to about 10 μM.

In some embodiments, the first culture medium comprises from 2 μM toabout 10 μM of CHIR-99021. In some embodiments, the second culturemedium comprises 50 ng/ml VEGF and 10 ng/ml bFGF. In other embodiments,the second culture medium further comprises Y-27632 and SB-431542. Invarious embodiments, the third culture medium comprises 10 μM Y-27632and 1 μM SB-431542. In certain embodiments, the third culture mediumfurther comprises VEGF and bFGF. In particular instances, the firstculture medium and/or the second medium is absent of insulin.

In some aspects, provided herein is an isolated, engineered O−hypoimmune dopaminergic neuron (DN) differentiated from a HIPO− cell,wherein endogenous 13-2 microglobulin (B2M) gene activity and endogenousclass II transactivator (CIITA) gene activity have been eliminated, CD47expression has been increased, and the neuron is blood type O and Rh−.

In some embodiments, the isolated O− hypoimmune dopaminergic neuron isselected from the group consisting of a neuronal stem cell, neuronalprogenitor cell, immature dopaminergic neuron, and mature dopaminergicneuron.

In some aspects, provided herein is a method of treating a patientsuffering from a neurodegenerative disease or condition. In someembodiments, the method comprises administering a composition comprisinga therapeutically effective amount of a population of any one of theisolated hypoimmune dopaminergic neurons. In some embodiments, thecomposition further comprises a therapeutically effective carrier. Insome embodiments, the population of the isolated hypoimmune dopaminergicneurons is on a biodegradable scaffold. In some embodiments, theadministration may comprise transplantation or injection. In someembodiments, the neurodegenerative disease or condition is selected fromthe group consisting of Parkinson's disease, Huntington disease, andmultiple sclerosis.

In some aspects, provided herein is a method of producing a populationof O− hypoimmune dopaminergic neurons from a population of HIPO− cellsby in vitro differentiation, wherein endogenous β-2 microglobulin (B2M)gene activity and endogenous class II transactivator (CIITA) geneactivity have been eliminated, CD47 expression has been increased, theblood group is O and Rh− in the HIPO-cells. In some embodiments, themethod comprises (a) culturing the population of HIPO-cells in a firstculture medium comprising one or more factors selected from the groupconsisting of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF8, WNT1,retinoic acid, a GSK3β inhibitor, an ALK inhibitor, and a ROCK inhibitorto produce a population of immature dopaminergic neurons; and (b)culturing the population of immature dopaminergic neurons in a secondculture medium that is different than the first culture medium toproduce a population of dopaminergic neurons.

In some embodiments, the GSKβ inhibitor is CHIR-99021, a derivativethereof, or a variant thereof. In some instances, the GSKβ inhibitor isat a concentration ranging from about 2 μM to about 10 μM. In someembodiments, the ALK inhibitor is SB-431542, a derivative thereof, or avariant thereof. In some instances, the ALK inhibitor is at aconcentration ranging from about 1 μM to about 10 μM. In someembodiments, the first culture medium and/or second culture medium areabsent of animal serum.

In some embodiments, the method also comprises isolating the populationof hypoimmune dopaminergic neurons from non-dopaminergic neurons. Insome embodiments, the method further comprises cryopreserving theisolated population of hypoimmune dopaminergic neurons.

In some aspects, provided herein is an isolated engineered O− hypoimmunepancreatic islet cell differentiated from a HIPO− cell, whereinendogenous β-2 microglobulin (B2M) gene activity and endogenous class IItransactivator (CIITA) gene activity have been eliminated, CD47expression has been increased, the blood type is O and Rh−.

In some embodiments, the isolated O− hypoimmune pancreatic islet cell isselected from the group consisting of a pancreatic islet progenitorcell, immature pancreatic islet cell, and mature pancreatic islet cell.

In some aspects, provided herein is a method of treating a patientsuffering from diabetes. The method comprises administering acomposition comprising a therapeutically effective amount of apopulation of any one of the isolated O− hypoimmune pancreatic isletcells described herein. In some embodiments, the composition furthercomprises a therapeutically effective carrier. In some embodiments, thepopulation of the isolated hypoimmune pancreatic islet cells is on abiodegradable scaffold. In some instances, the administration comprisestransplantation or injection.

In some aspects, provided herein is a method of producing a populationof O− hypoimmune pancreatic islet cells from a population of HIPO− cellsby in vitro differentiation, wherein endogenous β-2 microglobulin (B2M)gene activity and endogenous class II transactivator (CIITA) geneactivity have been eliminated, CD47 expression has been increased, theblood type is O and Rh− in the HIPO− cells. The method comprises: (a)culturing the population of HIPO-cells in a first culture mediumcomprising one or more factors selected from the group consistinginsulin-like growth factor (IGF), transforming growth factor (TGF),fibroblast growth factor (EGF), epidermal growth factor (EGF),hepatocyte growth factor (HGF), sonic hedgehog (SHH), and vascularendothelial growth factor (VEGF), transforming growth factor-β (TGFβ)superfamily, bone morphogenic protein-2 (BMP2), bone morphogenicprotein-7 (BMP7), a GSK3β inhibitor, an ALK inhibitor, a BMP type 1receptor inhibitor, and retinoic acid to produce a population ofimmature pancreatic islet cells; and (b) culturing the population ofimmature pancreatic islet cells in a second culture medium that isdifferent than the first culture medium to produce a population ofhypoimmune pancreatic islet cells.

In some embodiments, the GSK inhibitor is CHIR-99021, a derivativethereof, or a variant thereof. In some instances, the GSK inhibitor isat a concentration ranging from about 2 μM to about 10 μM. In someembodiments, the ALK inhibitor is SB-431542, a derivative thereof, or avariant thereof. In some instances, the ALK inhibitor is at aconcentration ranging from about 1 μM to about 10 μM. In someembodiments, the first culture medium and/or second culture medium areabsent of animal serum.

In some embodiments, the method also comprises isolating the populationof hypoimmune pancreatic islet cells from non-pancreatic islet cells. Insome embodiments, the method further comprises cryopreserving theisolated population of hypoimmune pancreatic islet cells.

In some aspects, provided herein is an isolated, engineered O−hypoimmune retinal pigmented epithelium (RPE) cell differentiated from aHIPO− cell, wherein endogenous 13-2 microglobulin (B2M) gene activityand endogenous class II transactivator (CIITA) gene activity have beeneliminated, CD47 expression has been increased, the blood type is O andRh−.

In some embodiments, the isolated O− hypoimmune RPE cell is selectedfrom the group consisting of an RPE progenitor cell, immature RPE cell,mature RPE cell, and functional RPE cell.

In some aspects, provided herein is a method of treating a patientsuffering from an ocular condition. The method comprises administering acomposition comprising a therapeutically effective amount of apopulation of any one of a population of the isolated hypoimmune RPEcells described herein. In some embodiments, the composition furthercomprises a therapeutically effective carrier. In some embodiments, thepopulation of the isolated hypoimmune RPE cells is on a biodegradablescaffold. In some embodiments, the administration comprisestransplantation or injection to the patient's retina. In someembodiments, the ocular condition is selected from the group consistingof wet macular degeneration, dry macular degeneration, juvenile maculardegeneration, Leber's Congenital Ameurosis, retinitis pigmentosa, andretinal detachment.

In some aspects, provided herein is a method of producing a populationof O− hypoimmune retinal pigmented epithelium (RPE) cells from apopulation of HIPO− cells) by in vitro differentiation, whereinendogenous β-2 microglobulin (B2M) gene activity and endogenous class IItransactivator (CIITA) gene activity have been eliminated and CD47expression has been increased in the HIPO-cells. The method comprises:(a) culturing the population of HIPO-cells in a first culture mediumcomprising any one of the factors selected from the group consisting ofactivin A, bFGF, BMP4/7, DKK1, IGF1, noggin, a BMP inhibitor, an ALKinhibitor, a ROCK inhibitor, and a VEGFR inhibitor to produce apopulation of pre-RPE cells; and (b) culturing the population of pre-RPEcells in a second culture medium that is different than the firstculture medium to produce a population of hypoimmune RPE cells.

In some embodiments, the ALK inhibitor is SB-431542, a derivativethereof, or a variant thereof. In some instances, the ALK inhibitor isat a concentration ranging from about 2 μM to about 10 μM. In someembodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or avariant thereof. In some instances, the ROCK inhibitor is at aconcentration ranging from about 1 μM to about 10 μM.

In some embodiments, the first culture medium and/or second culturemedium are absent of animal serum.

In some embodiments, the method further comprises isolating thepopulation of O− hypoimmune RPE cells from non-RPE cells. In someembodiments, the method further comprises cryopreserving the isolatedpopulation of hypoimmune RPE cells.

In one aspect, human pluripotent stem cells (hiPSCO-s) are renderedhypoimmunogenic by a) the disruption of the B2M gene at each allele(e.g. B2M−/−), b) the disruption of the CIITA gene at each allele (e.g.CIITA−/−), and c) by the overexpression of the CD47 gene (CD47+, e.g.through introducing one or more additional copies of the CD47 gene oractivating the genomic gene). This renders the hiPSCO− population B2M−/−CIITA−/− CD47tg. In a preferred aspect, the cells are non-immunogenic.In another embodiment, the HIPO− cells are rendered non-immunogenicB2M−/− CIITA−/− CD47tg as described above but are further modified byincluding an inducible suicide gene that is induced to kill the cells invivo when required. In other aspects, HIPO− cells are created when theHIP cells are rendered blood type O by knocking out the ABO gene Exon 7or silencing the SLC14A1 (JK) gene and the cells are rendered Rh− byknocking out the C and E antigens of the Rh blood group system (RH), Kin the Kell system (KEL), Fya and Fy3 in the Duffy system (FY), Jkb inthe Kidd system (JK), or U and S in the MNS blood group system.

F. Maintenance of O− Pluripotent Cells

Once generated, the iPSCO−, ESCO−, and HIPO− cells of the invention canbe maintained in an undifferentiated state as is known for maintainingiPSCs. For example, HIPO− cells can be cultured on Matrigel usingculture media, which prevents differentiation and maintainspluripotency.

G. Differentiation of O− Pluripotent Cells

The invention provides iPSCO−, ESCO−, and HIPO− cells that aredifferentiated into different cell types for subsequent transplantationinto subjects. As will be appreciated by those in the art, the methodsfor differentiation depend on the desired cell type using knowntechniques. The cells are differentiated in suspension and then put intoa gel matrix form, such as matrigel, gelatin, or fibrin/thrombin formsto facilitate cell survival. Differentiation is assayed as is known inthe art, generally by evaluating the presence of cell-specific markers.

In some embodiments, the iPSCO−, ESCO−, and HIPO− cells of the inventionare differentiated into hepatocytes to address loss of the hepatocytefunctioning or cirrhosis of the liver. There are a number of techniquesthat can be used to differentiate iPSCO−, ESCO−, and HIPO− cells intohepatocytes; see for example Pettinato et al., doi:10.1038/spre32888,Snykers et al., Methods Mol Biol 698:305-314 (2011), Si-Tayeb et al,Hepatology 51:297-305 (2010) and Asgari et al., Stem Cell Rev (:493-504(2013), all of which are hereby expressly incorporated by reference intheir entirety and specifically for the methodologies and reagents fordifferentiation. Differentiation is assayed as is known in the art,generally by evaluating the presence of hepatocyte associated and/orspecific markers, including, but not limited to, albumin, alphafetoprotein, and fibrinogen. Differentiation can also be measuredfunctionally, such as the metabolization of ammonia, LDL storage anduptake, ICG uptake and release and glycogen storage.

In some embodiments, the iPSCO−, ESCO−, and HIPO− cells aredifferentiated into beta-like cells or islet organoids fortransplantation to address type I diabetes mellitus (T1DM). Cell systemsare a promising way to address T1DM, see, e.g., Ellis et al.,doi/10.1038/nrgastro.2017.93, incorporated herein by reference.Additionally, Pagliuca et al. reports on the successful differentiationof β-cells from hiPSCs (see doi/10.106/j.cell.2014.09.040, herebyincorporated by reference in its entirety and in particular for themethods and reagents outlined there for the large-scale production offunctional human β cells from human pluripotent stem cells).Furthermore, Vegas et al. shows the production of human β cells fromhuman pluripotent stem cells followed by encapsulation to avoid immunerejection by the host; (doi:10.1038/nm.4030, hereby incorporated byreference in its entirety and in particular for the methods and reagentsoutlined there for the large-scale production of functional human βcells from human pluripotent stem cells).

Differentiation is assayed as is known in the art, generally byevaluating the presence of β cell associated or specific markers,including but not limited to, insulin. Differentiation can also bemeasured functionally, such as measuring glucose metabolism, seegenerally Muraro et al, doi:10.1016/j.cels.2016.09.002, herebyincorporated by reference in its entirety, and specifically for thebiomarkers outlined there.

Once the differentiated iPSCO−, ESCO−, and HIPO− beta cells aregenerated, they can be transplanted (either as a cell suspension orwithin a gel matrix as discussed herein) into the portal vein/liver, theomentum, the gastrointestinal mucosa, the bone marrow, a muscle, orsubcutaneous pouches.

In some embodiments, the iPSCO−, ESCO−, and HIPO− cells aredifferentiated into retinal pigment epithelium (RPE) to addresssight-threatening diseases of the eye. Human pluripotent stem cells havebeen differentiated into RPE cells using the techniques outlined inKamao et al., Stem Cell Reports 2014:2:205-18, hereby incorporated byreference in its entirety and in particular for the methods and reagentsoutlined there for the differentiation techniques and reagents; see alsoMandai et al., doi:10.1056/NEJMoa1608368, also incorporated in itsentirety for techniques for generating sheets of RPE cells andtransplantation into patients.

Differentiation can be assayed as is known in the art, generally byevaluating the presence of RPE associated and/or specific markers or bymeasuring functionally. See for example Kamao et al.,doi:10.1016/j.stemcr.2013.12.007, hereby incorporated by reference inits entirety and specifically for the markers outlined in the firstparagraph of the results section.

In some embodiments, the iPSCO−, ESCO−, and HIPO− cells of the inventionare differentiated into cardiomyocytes to address cardiovasculardiseases. Techniques are known in the art for the differentiation ofhiPSCs to cardiomyoctes and discussed in the Examples. Differentiationcan be assayed as is known in the art, generally by evaluating thepresence of cardiomyocyte associated or specific markers or by measuringfunctionally; see for example Loh et al.,doi:10.1016/j.cell.2016.06.001, hereby incorporated by reference in itsentirety and specifically for the methods of differentiating stem cellsincluding cardiomyocytes.

In some embodiments, the iPSCO−, ESCO−, and HIPO− cells aredifferentiated into endothelial colony forming cells (ECFCs) to form newblood vessels to address peripheral arterial disease. Techniques todifferentiate endothelial cells are known. See, e.g., Prasain et al.,doi:10.1038/nbt.3048, incorporated by reference in its entirety andspecifically for the methods and reagents for the generation ofendothelial cells from human pluripotent stem cells, and also fortransplantation techniques. Differentiation can be assayed as is knownin the art, generally by evaluating the presence of endothelial cellassociated or specific markers or by measuring functionally.

In some embodiments, the iPSCO−, ESCO−, and HIPO− cells aredifferentiated into thyroid progenitor cells and thyroid follicularorganoids that can secrete thyroid hormones to address autoimmunethyroiditis. Techniques to differentiate thyroid cells are known theart. See, e.g. Kurmann et al., doi:10.106/j.stem.2015.09.004, herebyexpressly incorporated by reference in its entirety and specifically forthe methods and reagents for the generation of thyroid cells from humanpluripotent stem cells, and also for transplantation techniques.Differentiation can be assayed as is known in the art, generally byevaluating the presence of thyroid cell associated or specific markersor by measuring functionally.

H. Transplantation of Differentiated HIPO− Cells

As will be appreciated by those in the art, the differentiated HIPO−derivatives are transplated using techniques known in the art thatdepends on both the cell type and the ultimate use of these cells. Ingeneral, the differentiated iPSCO−, ESCO−, and HIPO− cells of theinvention are transplanted either intravenously or by injection atparticular locations in the patient. When transplanted at particularlocations, the cells may be suspended in a gel matrix to preventdispersion while they take hold.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

VIII. EXAMPLES Example 1: Generation of Human iPSCs

The Human Episomal iPSC Line was derived from CD34+ cord blood (Cat. No.A33124, Thermo Fisher Scientific) using a three-plasmid, seven-factor(SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L Tantigen) EBNA-based episomal system from ThermoFisher. This iPSC line isconsidered to have a zero footprint as there was no integration into thegenome from the reprogramming event. It has been shown to be free of allreprogramming genes. The iPSCs have a normal XX karyotype and endogenousexpression of pluripotent markers like OCT4, SOX2, NANOG (as shown byRT-PCR) OCT4, SSEA4, TRA-1-60 and TRA-1-81 (as shown by ICC). Indirected differentiation and teratoma analyses, these hiPSCs retainedtheir differentiation potential for the ectodermal, endodermal, andmesodermal lineages. In addition, vascular, endothelial, and cardiaclineages were derived with robust efficiencies.

Several gene-delivery vehicles for iPSC generation were successfullyused, including retroviral vectors, adenoviral vectors, Sendai virus aswell as virus-free reprogramming methods (using episomal vectors,piggyBac transposon, synthetic mRNAs, microRNAs, recombinant proteins,and small molecule drugs, etc).

Different factors were successfully used for re-programming, such as thefirst reported combination of OCT3/4, SOX2, KLF4, and C-MYC, known asthe Yamanaka factors. In one embodiment, only three of these factorswere successfully combined and omitted C-MYC, although with reducedreprogramming efficiency.

In one embodiment, L-MYC or GLIS1 instead of C-MYC showed improvedreprogramming efficiency. In another embodiment, reprogramming factorsare not limited to genes associated with pluripotency.

All data are expressed as mean±SD or in box blot graphs showing themedian and the minimum to maximum range. Intergroup differences wereappropriately assessed by either the unpaired Student's t test or theone-way analysis of variance (ANOVA) with Bonferroni's postHoc test. *p<0.05, ** p<0.01.

Example 2: Generation of Human HIP Cells

Hypoimmune pluripotent (HIP) cells were generated as disclosed inWO2018/132783 and U.S. Prov. App. Nos. 62/698,941, 62/698,965,62/698,973, 62/698,978, 62/698,981, and 62/698,984, each of which areincorporated by reference herein in their entirety. Human Cas9 iPSCunderwent 2 gene-editing steps. In the first step, CRISPR technology wasperformed by a combined targeting of the coding sequence of humanbeta-2-microglobuline (B2M) gene with the CRISPR sequence5′-CGTGAGTAAACCTGAATCTT-3′ and the coding sequence of human CIITA genewith the CRISPR sequence 5′-GATATTGGCATAAGCCTCCC-3′. Linearized CRISPRsequence with T7 promoter was used to synthesize gRNA as per the kit'sinstructions (MEGAshortscript T7 Transcription Kit, Thermo Fisher). Thecollected in-vitro transcription (IVT) gRNA was then purified via theMEGAclear Transcription Clean-Up Kit. For IVT gRNA delivery,singularized cells were electroporated with 300 ng IVT gRNA using a Neonelectroporation system. After electroporation, edited Cas9 iPSCs wereexpanded for single cell seeding: iPSC cultures were dissociated tosingle cells using TrypLE (Gibco) and stained with Tra1-60 Alexa Fluor®488 and propidium iodide (PI). FACS Aria cell sorter (BD Biosciences)was used for the sorting and doublets and debris were excluded fromseeding by selective gating on forward and side scatter emission. Viablepluripotent cells were selected on the absence of PI and presence ofTra1-60 Alexa Fluor 488 staining. Single cells were then expanded intofull size colonies, after which the colonies were tested for a CRISPRedit. CRISPR mediated cleavage was assessed using the GeneArt GenomicCleavage Detection Kit (Thermo Fisher). Genomic DNA was isolated from1×10⁶ hiPSCs and the B2M and CIITA genomic DNA regions were PCRamplified using AmpliTaq Gold 360 Master Mix and the primer sets F:5′-TGGGGCCAAATCATGTAGACTC-3′ and R: 5′-TCAGTGGGGGTGAATTCAGTGT-3′ for B2Mas well as F: 5′-CTTAACAGCGATGCTGACCCC-3′ and R:5′-TGGCCTCCATCTCCCCTCTCTT-3′ for CIITA. For TIDE analysis, the obtainedPCR product was cleaned up (PureLink PCR Purification Kit, ThermoFisher) and Sanger sequencing was performed for the prediction of indelfrequency. After the confirmation of B2M/CIITA knockout, cells werefurther characterized through karyotype analysis and the TaqMan hPSCScorecard Panel (Thermo Fisher). The PSC were found to be pluripotentand maintained a normal (46, XX) karyotype during the genome editingprocess.

In the second step, the CD47 gene was synthesized and the DNA was clonedinto a plasmid lentivirus with an EF1a promotor and puromycinresistance. Cells were transduced with lentiviral stocks of 1×10⁷ TU/mLand 6 μg/mL of Polybrene (Thermo Fisher). Media was changed daily aftertransduction. Three days after transduction, cells were expanded andselected with 0.5 μg/mL of puromycin. After 5 days of antibioticselection, antibiotic resistant colonies emerged and were furtherexpanded to generate stable pools. Level of CD47 was confirmed by qPCR.Pluripotency assay (TaqMan hPSC Scorecard Panel, Thermo Fisher). andkaryotyping were performed again to verify the pluripotent status of thecells.

Example 3: HIP Cell Rejection in Rhesus Macaque Monkeys and Pigs

10 million hypoimmunogenic B2M−/− CIITA−/− rhesus CD47 tg iPSC derivedendothelial cells (expressing luciferase) were injected subcutaneouslyinto rhesus macaque monkeys and cells were longitudinally followed usingbioluminescence imaging; each animal was injected intravenously with 100mg/ml D-luciferin (Perkin Elmer, San Jose, Calif.) via a peripheralvessel for in vivo imaging using a Xenogen IVIS® 200 Series imagingsystem (Caliper Life Sciences, Alameda, Calif., Cat. No. 122799). Ourpre-transplant screening assays and validation studies predicted thatthe cells would not be rejected, resulting in stable BLI signals. TheBLI-signals decreased, however, on day 6 and were not detected by day16. In addition, “bumps” were observed on the injection sites (data notshown). Blood was drawn from the same monkeys and T cell, cytotoxic Tcell, NK cell (FIG. 1A), B cell (DSA; donor specific antibodies), ormacrophages activation (FIG. 1B) was not observed. Blood typing themonkeys by PCR confirmed that all tested monkeys were blood type B.Therefore, the hypoimmunogenic endothelial cell transplantation was ABOmismatched (data not shown). In contrast to these results, transplatingunmodified human iPSC-derived endothelial cells into Macaque Rhesusmonkeys resulted in rejection of the cells and the activation of anadaptive immune response (FIG. 2A). Additionally, transplanting HLA-Ideficient/HLA-II deficient B2M−/− CIITA−/− human iPSC-derivedendothelial cells into monkeys resulted in rejection of the cells andthe activation of an innate immune response (FIG. 2B).

Example 4: IgM Antibodies Killed Endothelial Cells from hiPSCs

Blood type rejection was confirmed by incubating human hypoimmunogenicendothelial cells with rhesus macaque serum.

Human iPSC differentiation into hiECs. hiPSC were plated on dilutedMatrigel (Corning, Tewksbury, Mass., Cat. No. 356231) in six-well platesand maintained in Essential 8 Flex media (Thermo Fisher, Cat. No.A2858501). The differentiation was started at 60% confluency and mediawas changed to RPMI-1640 containing 2% B-27 minus insulin (both GibcoThermo Fisher, Cat. No. A1895601) and 5 μM CHIR-99021 (Selleckchem, Cat.No. S1263). On day 2, the media was changed to reduced media: RPMI-1640containing 2% B-27 minus insulin and 2 μM CHIR-99021. From day 4 to 7,cells were exposed to RPMI-1640 EC media (RPMI-1640, 2% B-27 minusinsulin, 50 ng ml/l human vascular endothelial growth factor (VEGF,Peprotech, Rocky Hill, N.J., Cat. No. 100-20), 10 ng ml⁻¹ humanfibroblast growth factor basic (FGFb; Peprotech, Cat. No. 100-18B), 10μM Y-27632 (Selleckchem, Cat. No. S1049), and 1 μM SB 431542(Reagentsdirect, Cat. No. 21-A94).

Endothelial cell clusters were visible from day 7 and cells weremaintained in EGM-2 SingleQuots media (Lonza, Basel, Switzerland, Cat.No. CC-3162) plus 10% Fetal Calf Serum hi (Gibco Thermo Fisher, Cat. No.10082147), 25 ng ml−1 VEGF, 2 ng ml⁻¹ FGFb, 10 μM Y-27632 and 1 μM SB431542. The differentiation process was completed after 14 days andundifferentiated cells detached during the differentiation process. Forpurification, cells were treated with 20 μM PluriSln-1 (StemCellTechnologies, Cambridge, Mass., Cat. No. 72824) for 48 hours. The highlypurified ECs were cultured in EGM-2 SingleQuots media plus supplementsand 10% FCS hi (Gibco). TrypLE Express was used for passaging the cells1:3 every 3-4 days.

When human hypoimmunogenic B2M−/− CIITA−/− rhesus CD47 tg endothelialcells (blood type A) were incubated with rhesus macaque serum (bloodtype B), cells were killed immediately. The antibody type that killedthe cells was determined by antibody depletion analyses.

IgM depletion was done in a working solution of 50 mM dithiothreitol(DTT, Millipore Sigma, St. Louis, Mo., Cat. No. D0632). 10 μl of DTTwere mixed with 90 μl serum.

IgG depletion was done with Pierce Protein Beads (Thermo Fisher,Waltham, Mass., Cat. No. 88803). The beads were washed in the washbuffer and collected magnetically. 0.5 mg of the washed beads werecombined with 100 μl of the rhesus macaque serum and incubated for 60minutes at room temperature with gentle inversion every 10 minutes. Thebeads were separated magnetically, the serum was transferred to a newtube and kept on ice until used.

Depletion of either IgM or IgG antibodies demonstrated that theABO-antibodies are from the IgM type (FIG. 3). Blood type rejection wasalso similarly confirmed by IgM and IgG depletion using HIP-derivedcardiomyocytes and adult cardiac tissue (Celprogen, Torrance, Calif.,Cat. No. 36044-15-T75, data not shown).

Rhesus Monkey Blood Type B Serum Causes Complement-DependentCytotoxicity (CDC) of human wt iECs of blood type A. Human wt iPSCs ofblood type A or O were differentiated to wt iECs and incubated withserum from a rhesus monkey of blood type B on the XCelligence Real-TimeCell Analysis (ACEA Biosciences, San Diego, Calif.) platform.Differentiated iECs of blood type A were quickly killed (FIG. 2C)whereas iECs of blood type O were unaffected (FIG. 2D). Since anti-Aantibodies had been detected in this rhesus monkey 1 (using aclinically-approved agglutination test), iECs carrying the A blood typeantigen were killed and iECs not expressing the A antigen survived.

Embryonic stem cell-derived ECs undergo the same ABO bloodtype-dependent CDC as iPSC-derived iECs. To confirm that the ABO bloodtype sensitivity seen in iPSC-derived iECs is not inherent to the iPSCstarter cell, the H9 human embryonic stem cell line was used. ECs weredifferentiated from H9 which has blood type A, Rh+. Similar to theobservations with iPSC-derived iECs, H9-derived ECs underwent CDCkilling on the XCelligence platform when incubated with humanABO-incompatible serum (FIG. 2E) and rhesus monkey ABO-incompatibleserum (data not shown).

Example 5: Human HIP-Derived Cardiomyocytes or Mature CardiomyocytesSurvive Blood Group-compatible Xenogeneic serum Exposure

The human cells were not rejected by other pre-formed antibodies inxenogeneic transplantation. Human B2M−/− CIITA−/− rhesus CD47 tg-derivedendothelial cells (blood type A) were killed when incubated withABO-incompatible rhesus macaque serum (blood type B). When compatibleserum from rhesus macaque with blood type AB was used, however, thehuman cells survived (FIG. 4). Thus, rhesus macaques do not have otherpre-existing antibodies against human cells.

Human iPSCs were differentiated into hiCMs. hiPSCs were plated ondiluted Matrigel in six-well plates and maintained in Essential 8 Flexmedia (Thermo Fisher). Differentiation was started at 90% confluency,and media was changed to 5 ml of RPMI-1640 containing 2% B-27 minusInsulin and 6 μM CHIR-99021. After 2 days, media was changed toRPMI-1640 containing 2% B-27 minus insulin without CHIR. On day 3, 5 μlIWR1 (Selleckchem, Houston, Tex., Cat. No. S7086) was added to the mediafor two further days. At day 5, the media was changed back to RPMI-1640containing 2% B-27 minus insulin medium and left for 48 h. At day 7,media was changed to RPMI-1640 containing B27 plus insulin and replacedevery 3 days thereafter with the same media. Spontaneous beating ofcardiomyocytes was first visible around day 10. Purification ofcardiomyocytes was performed on day 10 post-differentiation. Briefly,media was changed to low glucose media and maintained for 3 days. At day13, media was changed back to RPMI-1640 containing B27 plus insulin.This procedure was repeated on day 14.

The human hypoimmunogenic iPSC-derived cardiomyocytes (blood type A)survive when incubated with allogeneic human serum blood type A and AB.However, serum containing pre-formed antibodies against A (blood type Oand B) killed the cells immediately (FIG. 5A). These results werevalidated using mature cardiomyocytes. Mature cardiomyocytes (purchasedfrom CELPROGEN) (blood type A) survived when incubated with allogeneicABO matched human serum (blood type A and AB), but they were killed whenincubated with serum containing pre-formed antibodies against A (bloodtype O and B) (FIG. 5b ).

Translational analyses were performed by incubating pig serum (bloodtype A) with human endothelial cells from all blood types. Only thosecells with blood type B or AB were killed (data not shown). Thisconfirms that human cells are hyperacutely rejected when transplantedinto ABO mismatched pigs.

Example 6: Hepatocytes Fail to Survive ABO Blood Group-incompatibleSerum Exposure

Human hepatocytes (blood type A; Corning, cat. No. 454543) survived whenincubated with ABO matched human serum (blood type A and AB) but thecells were killed when incubated with ABO-incompatible human serum(blood type B and O; BioChemed, Winchester, Va.), as measured usingXCelligence Real-Time Cell Analysis (ACEA Biosciences) to monitor cellsurvival/killing (FIG. 6). Similarly, human hepatocytes, which are bloodtype AB, survived when incubated with ABO matched human serum (bloodtype AB) but the cells were killed when incubated with ABO-incompatiblehuman serum (blood type A, B, and O).

This example demonstrates the importance of ABO matching in mature anddifferentiated hepatocytes.

Example 7: Relevance of Rhesus Factor in Sensitized Serum

Endothelial cells (blood type A Rh+ or blood type A Rh− survived whenincubated with an ABO matched human serum (blood type A) even when theserum was mismatched for Rh (Rh+ or Rh−), as long as the serum had notbeen previously sensitized to the Rh factor antigen (FIG. 7A). However,when endothelial cells which are blood type A Rh+ or blood type B Rh+were incubated with serum which is ABO-compatible (blood type AB) but Rhmismatched (Rh−), and the serum had been previously sensitized to the Rhantigen factor and contains anti-Rh antibodies, the cells were killed(FIG. 7B). Hypoimmunogenic endothelial cells (B2M−/− CIITA−/− CD47 tg)(blood type A Rh+) were also killed when incubated with anABO-compatible serum (blood type AB) which is Rh− and had beenpreviously sensitized to the Rh factor antigen and contains anti-Rhantibodies. Moreover, endothelial cells which are blood type O Rh+ werekilled when using O Rh− serum which had been previously sensitized tothe Rh factor and contains anti-Rh antibodies (FIG. 7C). This exampledemonstrates that use of Rh− cells may be important for transplantationinto previously sensitized subjects.

Embryonic stem cell-derived ECs undergo the same Rh blood type-dependentCDC as iPSC-derived iECs and primary wt ECs. FIG. 7D shows that whenH9-derived ECs of blood type A Rh+ were incubated with ABO-compatibleserum containing Rh antibodies, they underwent CDC killing.

Blood type O Rh− ECs do not undergo ABO− or Rh-mediated CDC. Theestablished EC line HEK293, a cell line derived from human embryonickidney cells grown in tissue culture, is blood type O Rh−. FIG. 7E showsthat the HEK293 incubated with serum containing Rh antibodies did notundergo killing.

Example 8: Generation of Human HIPO− Cells

In some embodiments, HIPO− cells are generated using blood type O Rh−pluripotent stem cells as starter cells and following the HIP cellgeneration protocols described herein. Therefore, the HIP cells areHIPO− cells.

In other aspects, a HIPO− cell is generated from a non-universal bloodgroup iPSC, ESC or HIP cell. For example, a blood type B− embryonic oriPSC cell line is transformed into O− by generating a knock-out cellline in the ABO gene using CRISPR technology for targeting of the codingsequence (gene ID: 28; Ensembl:ENSG00000175164 MIM:110300). Therefore,CRISPR guide RNAs targeting the coding sequence of the B gene areligated into vectors containing the Cas9 expression cassette andsubsequently transfected into hiPSCs. Linearized CRISPR sequence with T7promoter are used to synthesize gRNA as per the kit's instructions(MEGAshortscript T7 Transcription Kit, Thermo Fisher). The resulting invitro transcription (IVT) gRNA is then purified via the MEGAclearTranscription Clean-Up Kit. For IVT gRNA delivery, cells areelectroporated with 300 ng IVT gRNA using a Neon electroporation systemusing 1,200 V, 30 ms, 1 pulse into hiPSC stably expressing Cas9.

After electroporation, edited hiPSC are expanded for single cellseeding: hiPSC cultures are dissociated into single cells using TrypLEExpress (Gibco) and stained with Alexa Fluor 488-conjugated anti-TRA-160mAb and propidium iodide. A FACSAria II cell sorter (BD Biosciences) isused for the sorting and doublets and debris are excluded from seedingby selective gating on forward and side light scatter properties. Viablepluripotent cells are selected on the absence of propidium iodide andpresence of Tra1-60 staining. Single cells are then expanded intofull-size colonies, after which the colonies are tested for CRISPRediting by sequencing.

CRISPR-mediated cleavage is assessed using the GeneArt Genomic CleavageDetection Kit (Thermo Fisher) for testing of the initial edited pools.For screening the isolated clones, genomic DNA is isolated from 1×10⁶hiPSCs and the B genomic DNA regions are PCR amplified using AmpliTaqGold 360 Master Mix. For TIDE analysis, the resulting PCR product iscleaned up (PureLink PCR Purification Kit, Thermo Fisher) and Sangersequencing is performed for the prediction of indel frequency. After theconfirmation of B disruption, cells are further characterized throughkaryotype analysis and the TaqMan hiPSC Scorecard Panel (Thermo Fisher).

Another example is using a O rh+ cell line and transforming this into aO rh− cell line by deleting RHAG (Rh-associated glycoprotein; ammoniumtransport; associated with RhD; chromosome 6p21-qter) using CRISP/Cas9technology as described above (RHAG gRNA sequence: CCAGTGGGGCACTATTGTAC).

Example 9: Differentiation of Human HIPO− Cells

1. Differentiation of hHIPO− Cells to Human Cardiomyocytes

This is done using a protocol adapted from Sharma et al., J. Vis Exp.2015 doi: 10.3791/52628, hereby incorporated by reference in itsentirety and specifically for the techniques to differentiate the cells.HIPO− cells are plated on diluted Matrigel (356231, Corning) in 6-wellplates and maintained in Essential 8 Flex media (Thermo Fisher). Afterthe cells arrive at 90% confluency, the differentiation is started andmedia is changed to 5 mL of RPMI1640 containing 2% B-27 minus Insulin(both Gibco) and 6 uM CHIR-99021 (Selleck Chem). After 2 days, media ischanged to RPMI1640 containing 2% B-27 minus Insulin without CHIR. Onday 3, 5 uL IWR1 is added to the media for two further days. At day 5,the media is changed back to RPMI 1640 containing 2% B-27 minus insulinmedium and incubated for 48 hr. At day 7, media is changed to RPMI 1640containing B27 plus insulin (Gibco) and replaced every 3 days thereafterwith the same media. Spontaneous beating of cardiomyocytes may first bevisible at approximately day 10 to day 12. Purification ofCardiomyocytes is performed on day 10 post-differentiation. Briefly,media is changed to low glucose media and maintained for 3 days. At day13, media is changed back to RPMI 1640 containing B27 plus insulin. Thisprocedure is repeated on day 14. The remaining cells are highly purifiedcardiomyocytes.

2. Differentiation of HIPO− Cells to Human Endothelial Cells

HIPO− cells are plated on diluted Matrigel (356231, Corning) in 6-wellplates and maintained in Essential 8 Flex media (Thermo Fisher). Afterthe cells arrive at 60% confluency, the differentiation is started andmedia is changed to RPMI1640 containing 2% B-27 minus Insulin (bothGibco) and 5 μM CHIR-99021 (Selleck Chem). On day 2, the media ischanged to reduced media: RPMI1640 containing 2% B-27 minus Insulin(both Gibco) and 2 μM CHIR-99021 (Selleck Chem). From day 4 to day 7,cells are exposed to RPMI EC media, RPMI1640 containing 2% B-27 minusInsulin plus 50 ng/mL vascular endothelial growth factor (VEGF; R&DSystems, Minneapolis, Minn., USA), 10 ng/mL fibroblast growth factorbasic (FGFb; R&D Systems), 10 μM Y-27632 (Sigma-Aldrich, Saint Louis,Mo., USA) and 1 μM SB 431542 (Sigma-Aldrich). Endothelial cell clustersare visible from day 7 and cells are maintained in EGM-2 SingleQuotsmedia (Lonza, Basel, Switzerland) plus 10% FCS hi (Gibco), 25 ng/mLvascular endothelial growth factor (VEGF; R&D Systems, Minneapolis,Minn., USA), 2 ng/mL fibroblast growth factor basic (FGFb; R&D Systems),10 μM Y-27632 (Sigma-Aldrich, Saint Louis, Mo., USA) and 1 μM SB 431542(Sigma-Aldrich). The differentiation process may be completed after 14days and undifferentiated cells detach during the differentiationprocess. For purification, cells go through MACS progress according tothe manufactures' protocol using CD31 microbeads (Miltenyi, Auburn,Calif.). The highly purified EC-cells are cultured in EGM-2 SingleQuotsmedia (Lonza, Basel, Switzerland) plus supplements and 10% FCS hi(Gibco). TrypLE was used for passaging the cells 1:3 every 3 to 4 days.

IX. Exemplary sequences: Human β-2-Microglobulin SEQ ID NO: 1MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC RVNHVTLSQPKIVKWDRDIHuman CIITA protein, 160 amino acid N-terminus SEQ ID NO: 2MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELDQYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMPAEVGQKSQKRPFPEE LPADLKHWKP Human CD47SEQ ID NO: 3 MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVEHerpes Simplex Virus Thimidine Kinase (HSV-tk) SEQ ID NO: 4MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN Escherichia coli Cytosine Deaminase (EC-CD)SEQ ID NO: 5 MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQRAWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYPNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQSRFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVRGGKVIASTQPAQTTVYLEQPEAIDYKR Truncated human Caspase 9 SEQ ID NO: 6GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

All publications and patent documents disclosed or referred to hereinare incorporated by reference in their entirety. The foregoingdescription has been presented only for purposes of illustration anddescription. This description is not intended to limit the invention tothe precise form disclosed. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed:
 1. A modified pluripotent cell or a cell derivedtherefrom, wherein the cell is ABO blood group type O and Rhesus factornegative (Rh−), wherein the cell has (a) a reduced or eliminated ABOblood group antigen selected from the group consisting of A1, A2, and B;and/or (b) has a reduced or eliminated Rh protein antigen expressionselected from the group consisting of Rh C antigen, Rh E antigen, Kell Kantigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkbantigen, MNS antigen U, and MNS antigen S.
 2. A modified pluripotentcell or a cell derived therefrom, comprising: (a) a modification thatreduces or eliminates antigenicity of one or more ABO blood groupantigens selected from the group consisting of A1, A2, and B renderingthe cell ABO blood group type O; and/or (b) a modification that reducesor eliminates antigenicity of one or more Rh antigens selected from thegroup consisting of Rh C antigen, Rh E antigen, Kell K antigen (KEL),Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, MNSantigen U, and MNS antigen S rendering the cell Rh−.
 3. The modifiedpluripotent cell of claim 1 or claim 2, wherein the cell is ahypoimmunogenic pluripotent (HIP) cell comprising: a. an endogenousMajor Histocompatibility Antigen Class I (HLA-1) function that isreduced when compared to an unmodified pluripotent cell; b. anendogenous Major Histocompatibility Antigen Class II (HLA-11) functionthat is reduced when compared to an unmodified pluripotent cell; and c.an increased CD47 function that reduces susceptibility to NK cellkilling.
 4. A modified pluripotent cell or a cell derived thereform,wherein the cell comprises modulated expression of one or more of HLA-Ihuman leukocyte antigens, HLA-II human leukocyte antigens, CD47, CCR5,CXCR4, NLRC5, CIITA, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, PD-L1,CTLA-4-Ig, CD47, Cl-inhibitor, IL-35, RFX-5, RFXAP, RFXANK, NFY-A,NFY-B, NFY-C, IRF-1, OX40, GITR, 4-1BB, CD28, B7-1, B7-2, ICOS, CD27,HVEM, SLAM, CD226, PD1, CTL4, LAG3, TIGIT, TIM3, CD160, BTLA, CD244,CD30, TLT, VISTA, B7-H3, PD-L2, LFA-1, CD2, CD58, ICAM-3, TCRA, TCRB,FOXP3, HELIOS, ST2, PCSK9, APOC3, CD200, FASLG, CLC21, MFGE8, SERPIN B9,TGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1,and/or IFNγR1 d39 relative to a wild-type stem cell, wherein the cell isABO blood group type O or Rhesus factor negative (Rh−).
 5. A modifiedpluripotent cell or a cell derived therefrom, wherein the cell is an O−hypoimmunogenic pluripotent (HIPO−) cell comprising: a. an endogenousMajor Histocompatibility Antigen Class I (HLA-I) function that isreduced when compared to an unmodified pluripotent cell; b. anendogenous Major Histocompatibility Antigen Class II (HLA-II) functionthat is reduced when compared to an unmodified pluripotent cell; c. anincreased CD47 function that reduces susceptibility to NK cell killing;d. an ABO blood group type O (O); and e. optionally, a Rhesus factornegative (Rh−) blood type.
 6. The modified pluripotent cell or cellderived therefrom of any one of claims 1-5, wherein the cell is selectedfrom O− induced pluripotent stem cells (iPSCO−), O− embryonic stem cells(ESCO−), O− endothelial cells, O− cardiomyocytes, O− hepatocytes, O−dopaminergic neurons, O− pancreatic islet cells, O− retinal pigmentendothelium cells, and other O− cell types used for transplantation andmedical therapies, including O− chimeric antigen receptor (O− CAR)cells.
 7. The modified pluripotent cell or cell derived therefrom ofclaim 6, wherein the O− CAR cell is an O− chimeric antigen receptor T(O− CAR-T) cell.
 8. The modified pluripotent cell or cell derivedtherefrom of any one of claims 1-7, wherein said ABO blood group type Oresults from a reduced ABO blood group protein expression.
 9. Themodified pluripotent cell or cell derived therefrom of claim 8, whereinsaid ABO blood group type O results from a disruption in human Exon 7 ofthe ABO gene.
 10. The modified pluripotent cell or cell derivedtherefrom any one of claims 1-7, wherein said ABO blood group type Oresults from an enzymatic modification of an ABO gene product on asurface of said cell.
 11. The modified pluripotent cell or cell derivedtherefrom of claim 10, wherein said enzymatic modification removes acarbohydrate from said ABO gene product.
 12. The modified pluripotentcell or cell derived therefrom of claim 10, wherein said enzymaticmodification removes a carbohydrate from an ABO A1 antigen, A2 antigen,and/or B antigen.
 13. The modified pluripotent cell or cell derivedtherefrom of any one of claims 1-12, wherein said Rh− results from areduced Rh protein expression.
 14. The modified pluripotent cell or cellderived therefrom of claim 13, wherein said Rh− results from adisruption in an Rh C antigen, an Rh E antigen, a Kell K antigen (KEL),a Duffy (FY) Fya antigen, a Duffy Fy3 antigen, a Kidd (JK) Jkb antigen,or a Kidd SLC14A1 gene.
 15. The modified pluripotent cell or cellderived therefrom of any one of claims 1-7, wherein said ABO blood grouptype is endogenously type O.
 16. The modified pluripotent cell or cellderived therefrom of any one of claims 1-7, wherein said Rh− blood typeis endogenously type Rh−.
 17. The modified pluripotent cell or cellderived therefrom of any one of claims 3-16, wherein said HLA-I functionis reduced by a reduction in expression of a β-2 microglobulin protein.18. The modified pluripotent cell or cell derived therefrom of claim 17,wherein a gene encoding said β-2 microglobulin protein is knocked out.19. The modified pluripotent cell or cell derived therefrom of any oneof claims 3-16, wherein said HLA-I function is reduced by a reduction inexpression of an HLA-A protein.
 20. The modified pluripotent cell orcell derived therefrom of claim 19, wherein a gene encoding said HLA-Aprotein is knocked out.
 21. The modified pluripotent cell or cellderived therefrom of any one of claims 3-16, wherein said HLA-I functionis reduced by a reduction in expression of an HLA-B.
 22. The modifiedpluripotent cell or cell derived therefrom of claim 21, wherein a geneencoding said HLA-B protein is knocked out.
 23. The modified pluripotentcell or cell derived therefrom of any one of claims 3-16, wherein saidHLA-I function is reduced by a reduction in expression of an HLA-Cprotein.
 24. The modified pluripotent cell or cell derived therefrom ofclaim 23, wherein a gene encoding said HLA-C protein is knocked out. 25.The modified pluripotent cell or cell derived therefrom of any one ofclaims 3-24, wherein said HIPO− cell does not comprise an HLA-Ifunction.
 26. The modified pluripotent cell or cell derived therefrom ofany one of claims 3-25, wherein said HLA-II function is reduced by areduction in expression of a CIITA protein.
 27. The modified pluripotentcell or cell derived therefrom of claim 26, wherein a gene encoding saidCIITA protein is knocked out.
 28. The modified pluripotent cell or cellderived therefrom of any one of claims 3-25, wherein said HLA-IIfunction is reduced by a reduction in expression of an HLA-DP protein.29. The modified pluripotent cell or cell derived therefrom of claim 28,wherein a gene encoding said HLA-DP protein is knocked out.
 30. Themodified pluripotent cell or cell derived therefrom of any one of claims3-25, wherein said HLA-II function is reduced by a reduction inexpression of an HLA-DR protein.
 31. The modified pluripotent cell orcell derived therefrom of claim 30, wherein a gene encoding said HLA-DRprotein is knocked out.
 32. The modified pluripotent cell or cellderived therefrom of any one of claims 3-25, wherein said HLA-IIfunction is reduced by a reduction in expression of an HLA-DQ protein.33. The modified pluripotent cell or cell derived therefrom of claim 32,wherein a gene encoding said HLA-DQ protein is knocked out.
 34. Themodified pluripotent cell or cell derived therefrom of any one of claims3-33, wherein said hypoimmunogenic pluripotent cell does not comprise anHLA-II function.
 35. The modified pluripotent cell or cell derivedtherefrom of any one of claims 3-34, wherein said reduced susceptibilityto NK cell killing is caused by an increased expression of a CD47protein.
 36. The modified pluripotent cell or cell derived therefrom ofclaim 35, wherein said increased CD47 protein expression results from amodification to an endogenous CD47 gene locus.
 37. The modifiedpluripotent cell or cell derived therefrom of claim 35, wherein saidincreased CD47 protein expression results from a CD47 transgene.
 38. Themodified pluripotent cell or cell derived therefrom of any one of claims35-37, wherein said CD47 protein has at least a 90% sequence identity toSEQ ID NO:3.
 39. The modified pluripotent cell or cell derived therefromof claim 38, wherein said CD47 protein has the sequence of SEQ ID NO:3.40. The modified pluripotent cell or cell derived therefrom of any oneof claims 3-39, further comprising a suicide gene that is activated by atrigger that causes said hypoimmunogenic pluripotent cell to die. 41.The modified pluripotent cell or cell derived therefrom of claim 40,wherein said suicide gene is a herpes simplex virus thymidine kinasegene (HSV-tk) and said trigger is ganciclovir.
 42. The modifiedpluripotent cell or cell derived therefrom of claim 41, wherein saidHSV-tk gene encodes a protein comprising at least a 90% sequenceidentity to SEQ ID NO:4.
 43. The modified pluripotent cell or cellderived therefrom of claim 42, wherein said HSV-tk gene encodes aprotein comprising the sequence of SEQ ID NO:4.
 44. The modifiedpluripotent cell or cell derived therefrom of claim 40, wherein saidsuicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) andsaid trigger is 5-fluorocytosine (5-FC).
 45. The modified pluripotentcell or cell derived therefrom of claim 44, wherein said EC-CD geneencodes a protein comprising at least a 90% sequence identity to SEQ IDNO:5.
 46. The modified pluripotent cell or cell derived therefrom ofclaim 45, wherein said EC-CD gene encodes a protein comprising thesequence of SEQ ID NO:5.
 47. The modified pluripotent cell or cellderived therefrom of claim 40, wherein said suicide gene encodes aninducible Caspase protein and said trigger is a chemical inducer ofdimerization (CID).
 48. The modified pluripotent cell or cell derivedtherefrom of claim 47, wherein said gene encodes an inducible Caspaseprotein comprising at least a 90% sequence identity to SEQ ID NO:6. 49.The modified pluripotent cell or cell derived therefrom of claim 48,wherein said gene encodes an inducible Caspase protein comprising thesequence of SEQ ID NO:6.
 50. The modified pluripotent cell or cellderived therefrom of any one of claims 47-49, wherein said CID isAP1903.
 51. A cell derived from the modified pluripotent cell of any oneof claims 3-50, wherein said cell is selected from the group consistingof an O− chimeric antigen receptor (O− CAR) cell, an endothelial cell, adopaminergic neuron, a cardiac cell, a pancreatic islet cell, and aretinal pigment endothelium cell.
 52. The cell of claim 51, wherein saidO− CAR cell is an O− CAR-T cell.
 53. The modified pluripotent cell orcell derived therefrom of any one of claims 1-51, wherein the modifiedpluripotent cell or cell derived therefrom is a cardiomyocyte orcardiomyocyte progenitor cell.
 54. The modified pluripotent cell or cellderived therefrom of any one of claims 1-53 further comprising a reducedor eliminated expression of a CCR5 gene or a CXCR4 gene.
 55. Themodified pluripotent cell or cell derived therefrom of any one of claims1-54 further comprising a reduce or eliminated expression of an NLRC5gene.
 56. The modified pluripotent cell or cell derived therefrom of anyone of claims 1-55 further comprising a modified expression of at leastone of gene selected from the group consisting of HLA-A, HLA-B, HLA-C,HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, Cl-inhibitor, and IL-35.
 57. Themodified pluripotent cell or cell derived therefrom of any one of claims1-56 further comprising a modified expression of at least one geneselected from RFX-5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, and IRF-1. 58.The modified pluripotent cell or cell derived therefrom of any one ofclaims 1-57 further comprising a modified expression of at least onegene selected from the group consisting of OX40, GITR, 4-1BB, CD28,B7-1, B7-2, ICOS, CD27, HVEM, SLAM, CD226, PD1, CTL4, LAG3, TIGIT, TIM3,CD160, BTLA, CD244, CD30, TLT, VISTA, B7-H3, PD-L2, LFA-1, CD2, CD58,ICAM-3, TCRA, TCRB, FOXP3, HELIOS, ST2, PCSK9, CCR5, and APOC3.
 59. Themodified pluripotent cell or cell derived therefrom of any one of claims1-58 further comprising at least one transgene selected from the groupconsisting of PDL-1, HLA-G, CD47, CD200, FASLG, CLC21, MFGE8, and SERPINB9, or at least one transgene encoding an agonist of PDL-1, HLA-G, CD47,CD200, FASLG, CLC21, MFGE8, or SERPIN
 9. 60. The modified pluripotentcell or cell derived therefrom of any one of claims 1-59 furthercomprising at least one transgene selected from the group consisting ofTGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1,and IFNγR1 d39, or at least one transgene encoding an agonist of TGFβ,CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1, orIFNγR1 d39.
 61. A method of transplanting a cell, comprisingadministering one or more cells derived from the modified pluripotentcell of any one of claims 1-60 into a subject in need thereof, whereinsaid subject is a human, cow, pig, chicken, turkey, horse, sheep, goat,donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse,rat, dog, cat, hamster, guinea pig.
 62. The method of claim 61, whereinsaid cell derived from the modified pluripotent cell is selected fromthe group consisting of an O− CAR cell, an O− endothelial cell, an O−dopaminergic neuron, an O− cardiac cell, an O− pancreatic islet cell,and an O− retinal pigment endothelium cell.
 63. A method of treating adisease in a patient in need of transplanted cells, comprisingadministering to the patient a cell derived from the modifiedpluripotent cell of any one of claims 1-60.
 64. The method of claim 63,wherein said cell derived from the modified pluripotent cell is selectedfrom the group consisting of an O− CAR cell, an O− endothelial cell, anO− dopaminergic neuron, an O− cardiac cell, an O− pancreatic islet cell,and an O− retinal pigment endothelium cell.
 65. The method of claim 63,wherein said disease is selected from the group consisting of Type IDiabetes, a cardiac disease, a neurological disease, a cancer, an oculardisease, and a vascular disease.
 66. A method for generating a modifiedpluripotent cell that is ABO blood type O and Rh factor negative (Rh−)from a pluripotent cell that is not O−, comprising: a. eliminating orreducing the expression of any ABO blood group antigens A1, A2, and/or Bto provide an ABO blood type O; and/or b. eliminating or reducingexpression of the Rhesus factor (Rh) blood group antigen to provide ablood type Rh negative (−).
 67. A method for generating ahypoimmunogenic pluripotent ABO group O Rh factor negative (HIPO−) cellfrom an O− pluripotent cell comprising: a. eliminating the MajorHistocompatibility Antigen Class I (HLA-I) function when compared to anunmodified pluripotent cell; b. eliminating the Major HistocompatibilityAntigen Class II (HLA-I) function when compared to an unmodifiedpluripotent cell; and c. increasing the expression of CD47 as comparedto an unmodified pluripotent cell.
 68. The method of claim 66 or 67,wherein said pluripotent cell is of human, cow, pig, chicken, turkey,horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak,llama, alpaca, mouse, rat, dog, cat, hamster, or guinea pig origin. 69.The method of any one of claims 66-68, wherein said ABO blood group typeO results from eliminating an ABO blood group protein expression. 70.The method of any one of claims 66-69, wherein the cell is endogenouslytype O.
 71. The method of any one of claims 66-69, wherein thepluripotent cell is human and wherein the ABO blood group type O resultsfrom disrupting a human Exon 7 of the ABO gene.
 72. The method of claim71, wherein disrupting the human Exon 7 of the ABO gene is accomplishedby a Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/Cas9 reaction that disrupts both alleles of the Exon
 7. 73. Themethod of any one of claims 66-69, wherein said ABO blood group type Oresults from enzymatically modifying an ABO gene product on a surface ofthe pluripotent cell.
 74. The method of claim 73, wherein said enzymaticmodification removes a carbohydrate from said ABO gene product.
 75. Themethod of claim 74, wherein said enzymatic modification removes acarbohydrate from an ABO A1 antigen, A2 antigen, and/or B antigen. 76.The method of any one of claims 66-69, wherein the cell is endogenouslytype Rh−.
 77. The method of any one of claims 66-69, comprisingeliminating an Rh protein expression.
 78. The method of claim 77,wherein said type Rh− results from disrupting a gene encoding Rh Cantigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen,Duffy Fy3 antigen, Kidd (JK) Jkb antigen, or Kidd SLC14A1 gene.
 79. Themethod of claim 78, wherein said disruption results from a CRISPR/Cas9reaction that disrupts both alleles of said gene.
 80. The method of anyone of claims 67-79, wherein said increased CD47 expression results fromintroducing at least one copy of a human CD47 gene under the control ofa promoter into said parent cell.
 81. The method of claim 80, whereinsaid promoter is a constitutive promoter.
 82. The method of any one ofclaims 67-81, comprising disrupting both alleles of said B2M gene (e.g.,using a CRISPR/Cas9 reaction).
 83. The method of any one of claims67-82, wherein said HLA-I function is reduced by reducing expression ofa β-2 microglobulin protein.
 84. The method of claim 83, whereinexpression of the β-2 microglobulin protein is reduced by knocking out agene encoding said β-2 microglobulin protein.
 85. The method of claim84, wherein said β-2 microglobulin protein has at least a 90% sequenceidentity to SEQ ID NO:1.
 86. The method of claim 85, wherein said β-2microglobulin protein has the sequence of SEQ ID NO:1.
 87. The method ofany one of claims 67-81, wherein said HLA-I function is reduced byreducing expression of an HLA-A protein.
 88. The method of claim 87,wherein expression of said HLA-A protein is reduced by knocking out agene encoding said HLA-A protein.
 89. The method of any one of claims67-81, wherein said HLA-I function is reduced by reducing expression ofan HLA-B protein.
 90. The method of claim 89, wherein expression of saidHLA-B protein is reduced by knocking out a gene encoding said HLA-Bprotein.
 91. The method of any one of claims 67-81, wherein said HLA-Ifunction is reduced by reducing expression of an HLA-C protein.
 92. Themethod of claim 91, wherein expression of said HLA-C protein is reducedby knocking out a gene encoding said HLA-C protein.
 93. The method ofany one of claims 67-92, wherein said hypoimmunogenic pluripotent celldoes not comprise an HLA-I function.
 94. The method of any one of claims67-93, wherein said HLA-II function is reduced by reducing expression ofa CIITA protein.
 95. The method of claim 94, wherein expression of saidCIITA protein is reduced by knocking out a gene encoding said CIITAprotein.
 96. The method of claim 95, wherein said CIITA protein has atleast a 90% sequence identity to SEQ ID NO:2.
 97. The method of claim96, wherein said CIITA protein has the sequence of SEQ ID NO:2.
 98. Themethod of any one of claims 67-97, comprising reducing expression of aCIITA protein by a CRISPR reaction that disrupts both alleles of a CIITAgene.
 99. The method of any one of claims 67-98, wherein said HLA-IIfunction is reduced by reducing expression of an HLA-DP protein. 100.The method of claim 99, wherein expression of said HLA-DP protein isreduced by knocking out a gene encoding said HLA-DP protein.
 101. Themethod of any one of claims 67-98, wherein said HLA-II function isreduced by reducing expression of an HLA-DR protein.
 102. The method ofclaim 101, wherein expression of said HLA-DR protein is reduced byknocking out a gene encoding said HLA-DR protein.
 103. The method of anyone of claims 67-98, wherein said HLA-II function is reduced by reducingexpression of an HLA-DQ protein.
 104. The method of claim 103, whereinexpression of said HLA-DQ protein is reduced by knocking out a geneencoding said HLA-DQ protein.
 105. The method of any one of claims67-104, wherein said hypoimmunogenic pluripotent cell does not comprisean HLA-II function.
 106. The method of any one of claims 67-105, whereinsaid increased expression of CD47 results from expression of a transgenethat encodes a CD47 protein.
 107. The method of claim 106, wherein saidCD47 protein has at least a 90% sequence identity to SEQ ID NO:3. 108.The method of claim 107, wherein said CD47 protein has the sequence ofSEQ ID NO:3.
 109. The method of any one of claims 67-108, furthercomprising expressing a suicide gene that is activated by a trigger thatcauses said hypoimmunogenic pluripotent cell to die.
 110. The method ofclaim 109, wherein said suicide gene is a herpes simplex virus thymidinekinase gene (HSV-tk) and said trigger is ganciclovir.
 111. The method ofclaim 110, wherein said HSV-tk gene encodes a protein comprising atleast a 90% sequence identity to SEQ ID NO:4.
 112. The method of claim111, wherein said HSV-tk gene encodes a protein comprising the sequenceof SEQ ID NO:4.
 113. The method of claim 109, wherein said suicide geneis an Escherichia coli cytosine deaminase gene (EC-CD) and said triggeris 5-fluorocytosine (5-FC).
 114. The method of claim 113, wherein saidEC-CD gene encodes a protein comprising at least a 90% sequence identityto SEQ ID NO:5.
 115. The method of claim 114, wherein said EC-CD geneencodes a protein comprising the sequence of SEQ ID NO:5.
 116. Themethod of claim 109, wherein said suicide gene encodes an inducibleCaspase protein and said trigger is a specific chemical inducer ofdimerization (CID).
 117. The method of claim 116, wherein said geneencodes an inducible Caspase protein comprising at least a 90% sequenceidentity to SEQ ID NO:6.
 118. The method of claim 117, wherein said geneencodes an inducible Caspase protein comprising the sequence of SEQ IDNO:6.
 119. The method of any one of claims 116-118, wherein said CID isAP1903.
 120. A method of transplanting a hypoimmunogenic pluripotent(HIP) cell or cell derived therefrom into a subject in need thereofcomprising: determining an ABO blood group type of the HIP cell or cellderived therefrom, determining an ABO blood group type of the subject,and transplanting the HIP cell or cell derived therefrom only if the ABOblood group type of the HIP cell or cell derived therefrom matches theABO blood group type of the subject.
 121. The method of claim 120,further comprising: determining a Rhesus (Rh) factor type of the HIPcell or cell derived therefrom, determining an Rh factor type of thesubject, and transplanting the HIP cell or cell derived therefrom onlyif the Rh blood type of the HIP cell or cell derived therefrom matchesthe Rh blood type of the subject.
 122. The method of claim 120 or 121,wherein the HIP cell or cell derived therefrom comprises a. anendogenous Major Histocompatibility Antigen Class I (HLA-I) functionthat is reduced when compared to an unmodified pluripotent cell; b. anendogenous Major Histocompatibility Antigen Class II (HLA-II) functionthat is reduced when compared to an unmodified pluripotent cell; and c.an increased CD47 function that reduces susceptibility to NK cellkilling.
 123. The method of any one of claims 119-120, wherein the cellderived from the HIP cell is a cardiomyocyte or cardiomyocyteprogenitor.